Cyclic peptides as g-protein coupled receptor antagonists

ABSTRACT

The invention relates to novel cyclic compounds which have the ability to modulate the activity of G protein-coupled receptors. The compounds preferably act as antagonists. In preferred embodiments, the invention provides cyclic peptidic and peptidomimetic antagonists of C5a receptors, which are active against C5a receptors on polymorphonuclear leukocytes and macrophages. The compounds of the invention are both potent and selective, and are useful in the treatment of a variety of inflammatory conditions.

The present application is a continuation of U.S. application Ser. No.10/493,117, filed Oct. 24, 2005 (now allowed), which was the UnitedStates national stage of PCT International Patent ApplicationPCT/NZ2000/00064, filed Apr. 28, 2000 (now expired), which claimedpriority to New Zealand Provisional Application No. 335553, filed Apr.30, 1999 (now expired); the contents of each of which is specificallyincorporated herein in its entirety by express reference thereto.

FIELD OF THE INVENTION

The invention relates to novel cyclic compounds which have the abilityto modulate the activity of G protein-coupled receptors. The compoundspreferably act as antagonists. In preferred embodiments, the inventionprovides cyclic peptidic and peptidomimetic antagonists of C5areceptors, which are active against C5a receptors on polymorphonuclearleukocytes and macrophages. The compounds of the invention are bothpotent and selective, and are useful in the treatment of a variety ofinflammatory conditions. This invention relates to novel cycliccompounds which have the ability to modulate the activity of Gprotein-coupled receptors. The compounds preferably act as antagonists.In preferred embodiments, the invention provides cyclic peptidic andpeptidomimetic antagonists of C5a receptors, which are active againstC5a receptors on polymorphonuclear leukocytes and macrophages. Thecompounds of the invention are both potent and selective, and are usefulin the treatment of a variety of inflammatory conditions.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

G protein-coupled receptors are prevalent throughout the human body,comprising approximately 60% of known cellular receptor types, andmediate signal transduction across the cell membrane for a very widerange of endogenous ligands. They participate in a diverse array ofphysiological and pathophysiological processes, including, but notlimited to those associated with cardiovascular, central and peripheralnervous system, reproductive, metabolic, digestive, immunoinflammatory,and growth disorders, as well as other cell-regulatory and proliferativedisorders. Agents which selectively modulate functions of Gprotein-coupled receptors have important therapeutic applications. Thesereceptors are becoming increasingly recognised as important drugtargets, due to their crucial roles in signal transduction (Gprotein-coupled Receptors, IBC Biomedical Library Series, 1996).

One of the most intensively studied G protein-coupled receptors is thereceptor for C5a. C5a is one of the most potent chemotactic agentsknown, and recruits neutrophils and macrophages to sites of injury,alters their morphology; induces degranulation; increases calciummobilisation, vascular permeability (oedema) and neutrophiladhesiveness; contracts smooth muscle; stimulates release ofinflammatory mediators, including histamine, TNF-α, IL-1, IL-6, IL-8,prostaglandins, and leukotrienes, and of lysosomal enzymes; promotesformation of oxygen radicals; and enhances antibody production (Gerardand Gerard, 1994).

Overexpression or underregulation of C5a is implicated in thepathogenesis of immune system-mediated inflammatory conditions, such asrheumatoid arthritis, adult respiratory distress syndrome (ARDS),systemic lupus erythematosus, tissue graft rejection, ischaemic heartdisease, reperfusion injury, septic shock, psoriasis, gingivitis,atherosclerosis, Alzheimer's disease, lung injury and extracorporealpost-dialysis syndrome, and in a variety of other conditions (Whaley1987; Sim 1933).

Agents which limit the pro-inflammatory actions of C5a have potentialfor inhibiting chronic inflammation, and its accompanying pain andtissue damage. For these reasons, molecules which prevent C5a frombinding to its receptors are useful for treating chronic inflammatorydisorders driven by complement activation. Such compounds also providevaluable new insights into the mechanisms of complement-mediatedimmunity.

In our previous application No. PCT/AU98/00490, the entire disclosure ofwhich is incorporated herein by this reference, we described thethree-dimensional structure of some analogues of the C-terminus of humanC5a, and used this information to design novel compounds which bind tothe human C5a receptor (C5aR), behaving as either agonists orantagonists of C5a. It had previously been thought that a putativeantagonist might require both a C-terminal arginine and a C-terminalcarboxylate for receptor binding and antagonist activity (Konteatis etal, 1994). In PCT/AU98/00490, but we showed that in fact a terminalcarboxylate group is not generally required either for high affinitybinding to C5aR or for antagonist activity. Instead we found that ahitherto unrecognized structural feature, a turn conformation, was thekey recognition feature for high affinity binding to the human C5areceptor on neutrophils. We used these findings to design constrainedstructural templates which enable hydrophobic groups to be assembledinto a hydrophobic array for interaction with a C5a receptor.

By investigating the effect of varying the structure at each amino acidresidue in the most potent compound identified in our previousapplication, we have now developed further examples of cyclicantagonists of the C5a receptor on human neutrophils and have identifiedpotent C5aR antagonist activity for a range of compounds. Thesecompounds each comprise a cyclic scaffold which satisfies the generalthree-dimensional structural requirements set out in the earlierapplication No. PCT/AU98/00490, but we have now found that certainsubstituents attached to the cycle surprisingly lead to most unexpectedresults, producing both high and low antagonist potencies which were notaccurately predicted in the previous application No. PCT/AU98/00490.These surprising new findings allow us to refine and better define therequired pharmacophore for antagonism of C5a receptors. The unexpectedstructure-activity relationships described herein help to define arefined structural pharmacophore for active antagonism of C5a receptorson human polymorphonuclear leukocytes (neutrophil granulocytes). Thispharmacophore is expected to be appropriate also for C5a receptors onother human and mammalian cells.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a compound which isan antagonist of a G protein-coupled receptor, which has substantiallyno agonist activity, and which is a cyclic peptide or peptidomimetic offormula I:

where A is H, alkyl, aryl, NH₂, NH-alkyl, N(alkyl)₂, NH-aryl, NH-acyl,NH-benzoyl, NHSO₃, NHSO₂-alkyl, NHSO₂-aryl, OH, O-alkyl, or O-aryl;

B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or theside chain of a D- or L-amino acid such as L-phenylalanine orL-phenylglycine, but is not the side chain of glycine, D-phenylalanine,L-homophenylalanine, L-tryptophan, L-homotryptophan, L-tyrosine, orL-homotyrosine;

C is a small substituent, such as the side chain of a D-, L- orhomo-amino acid such as glycine, alanine, leucine, valine, proline,hydroxyproline, or thioproline, but is preferably not a bulkysubstituent such as isoleucine, phenylalanine, or cyclohexylalanine;

D is the side chain of a neutral D-amino acid such as D-Leucine,D-homoleucine, D-cyclohexylalanine, D-homocyclohexylalanine, D-valine,D-norleucine, D-homo-norleucine, P-phenylalanine,D-tetrahydroisoquinoline, D-glutamine, D-glutamate, or D-tyrosine, butis preferably not a small substituent such as the side chain of glycineor D-alanine, a bulky planar side chain such as D-tryptophan, or a bulkycharged side chain such as D-arginine or D-Lysine;

E is a bulky substituent, such as the side chain of an amino acidselected from the group consisting of L-phenylalanine, L-tryptophan andL-homotryptophan, or is L-1-napthyl or L-3-benzothienyl alanine, but isnot the side chain of D-tryptophan, L-N-methyltryptophan,L-homophenylalanine, L-2-naphthyl L-tetrahydroisoquinoline,L-cyclohexylalanine, D-leucine, L-fluorenylalanine, or L-histidine;

F is the side chain of L-arginine, L-homoarginine, L-citrulline, orL-canavanine, or a bioisostere thereof, i.e. a side chain in which theterminal guanidine or urea group is restrained, but the carbon backboneis replaced by a group which has different structure, but is such thatthe side chain as a whole reacts with the target protein in the same wayas the parent group;

X is —(CH₂)_(n)NH— or (CH₂)_(n)—S—, where n is an integer of from 1 to4, preferably 2 or 3; —(CH₂)₂O—; —(CH₂)₃O—; —(CH₂)₃—; —(CH₂)₄—;—CH₂COCHRNH—; or —CH₂—CHCOCHRNH—, and where R is the side chain of anycommon or uncommon amino acid, with the proviso that the compound is notcompound 1 referred to below.

In C, both the cis and trans forms of hydroxyproline and thioproline maybe used.

Preferably A is an acetamide group, an aminomethyl group, or asubstituted or unsubstituted sulphonamide group.

Preferably where A is a substituted sulphonamide, the substituent is analkyl chain of 1 to 6, preferably 1 to 4 carbon atoms, or a phenyl ortoluoyl group.

Preferably the G protein-coupled receptor is a C5a receptor. However, wehave found that the leading compound of our earlier application also hassignificant binding affinity at vasopressin and neurokinin receptors,and therefore these receptors are also within the scope of theinvention.

In a particularly preferred embodiment, the compound has antagonistactivity against C5aR, and has no C5a agonist activity.

The cyclic compounds of the invention are preferably antagonists of C5areceptors on human, mammalian cells including, but not limited to, humanpolymorphonuclear leukocytes and human macrophages. The compounds of theinvention preferably bind potently and selectively to C5a receptors, andmore preferably have potent antagonist activity at sub-micromolarconcentrations. Even more preferably the compound has a receptoraffinity IC₅₀<25 μM, and an antagonist potency IC₅₀<1 μM.

Still more preferably the compound is selected from the group consistingof compounds 2 to 6, 10 to 15, 17, 19, 20, 22, 25, 26, 28, 30, 31, 33 to37, 39 to 45, 47 to 50, 52 to 58 and 60 to 70 described herein. Mostpreferably the compound is compound 33, compound 60 or compound 45.

For the purposes of this specification, the term “alkyl” is to be takento mean a straight, branched, or cyclic, substituted or unsubstitutedalkyl chain of 1 to 6, preferably 1 to 4 carbons. Most preferably thealkyl group is a methyl group. The term “acyl” is to be taken to mean asubstituted or unsubstituted acyl of 1 to 6, preferably 1 to 4 carbonatoms. Most preferably the acyl group is acetyl. The term “aryl” is tobe understood to mean a substituted or unsubstituted homocyclic orheterocyclic aryl group, in which the ring preferably has 5 or 6members.

A “common” amino acid is a L-amino acid selected from the groupconsisting of glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate,glutamine, cysteine, methionine, arginine, lysine, proline, serine,threonine and histidine.

An “uncommon” amino acid includes, but is not restricted to, D-aminoacids, homo-amino acids, N-alkyl amino acids, dehydroamino acids,aromatic amino acids other than phenylalanine, tyrosine and tryptophan,ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline,canavanine, norleucine, δ-glutamic acid, aminobutyric acid,L-fluorenylalanine, L-3-benzothienylalanine, and α, α-disubstitutedamino acids.

According to a second aspect, the invention provides a compositioncomprising a compound according to the invention, together with apharmaceutically-acceptable carrier or excipient.

The compositions of the invention may be formulated for use in oral,parenteral, inhalational, intranasal, transdermal or other topicalapplications, but oral or topical formulations are preferred. Fortopical administration, vehicles such as dimethylsulphonate or propyleneglycol may be used. Other vehicles may be preferred depending on thetissue surface to be treated.

It is expected that most if not all compounds of the invention will bestable in the presence of metabolic enzymes such as those of the gut,blood, lung or intracellular enzymes. Such stability can readily betested by routine methods known to those skilled in the art.

Suitable formulations for administration by any desired route may beprepared by standard methods, for example by reference to well-knowntextbooks such as Remington: The Science and Practice of Pharmacy, Vol.II, 2000 (20th edition), A. R. Gennaro (ed), Williams & Wilkins,Pennsylvania.

In a third aspect, the invention provides a method of treatment of apathological condition mediated by a G protein-coupled receptor,comprising the step of administering an effective amount of a compoundof the invention to a mammal or vertebrate in need of such treatment.

Preferably the condition mediated by a G protein-coupled receptor is acondition mediated by a C5a receptor, and more preferably involvesoverexpression or underregulation under-regulation of C5a. Suchconditions include but are not limited to rheumatoid arthritis, adultrespiratory distress syndrome (ARDS), systemic lupus erythematosus,tissue graft rejection, ischaemic heart disease, reperfusion injury,septic shock, gingivitis, fibrosis, atherosclerosis, multiple sclerosis,Alzheimer's disease, asthma, dementias, central nervous systemdisorders, lung injury, extracorporeal post-dialysis syndrome, anddermal inflammatory disorders such as psoriasis, eczema and contactdermatitis.

In one preferred embodiment the condition is rheumatoid arthritis.

In a second preferred embodiment the condition is reperfusion injury. Inthis second embodiment it will be clearly understood that the proviso toformula I does not apply.

While the invention is not in any way restricted to the treatment of anyparticular animal or species, it is particularly contemplated that thecompounds of the invention will be useful in medical treatment ofhumans, and will also be useful in veterinary treatment, particularly ofcompanion animals such as cats and dogs, livestock such as cattle,horses and sheep, and zoo animals, including non-human primates, largebovids, felids, ungulates and canids. Other species which may beamenable to treatment include reptiles, fishes or amphibians.

The compounds may be administered at any suitable dose and by anysuitable route. Oral, topical, transdermal or intranasal administrationis preferred, because of the greater convenience and acceptability ofthese routes. Topical applications could also include the use offormulations such as pessaries or suppositories for vaginal or rectaladministration or the use of aqueous drops for topical administration toears or eyes. The effective dose will depend on the nature of thecondition to be treated, and the age, weight, and underlying state ofhealth of the individual treatment. This be at the discretion of theattending physician or veterinarian. Suitable dosage levels may readilybe determined by trial and error experimentation, using methods whichare well known in the art.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the inhibition of thevascular leakage associated with a dermal Arthus reaction by intravenous(FIG. 1A), oral (FIG. 1B) and topical (FIG. 1C) AcF-[OPdChaWR], andappropriate controls (FIG. 1D).

FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the inhibition of the risein circulating TNF-α associated with a dermal Arthus reaction byintravenous (FIG. 2A), oral (FIG. 2B) and topical (FIG. 2C)AcF-[OPdChaWR], and appropriate topical controls (FIG. 2D).

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show the reduction of thepathology index associated with a dermal Arthus reaction by intravenous,oral and topical AcF-[OPdChaWR].

FIG. 4 shows the effect of a C5a antagonist on gut ischemia-reperfusioninduced intestinal edema.

FIG. 5 shows the effect of a C5a antagonist on gut ischemia-reperfusioninduced neutropenia.

FIG. 6 shows the effect of a C5a antagonist on gut Ischemia-reperfusioninduced serum TNF-α elevation.

FIG. 7 shows the effect of a C5a antagonist on gut ischemia-reperfusioninduced serum haptoglobin elevation.

FIG. 8 shows the effect of a C5a antagonist on gut ischemia-reperfusioninduced aspartate aminotransferase.

FIG. 9 shows the effect of a C5a antagonist on histopathology of gutischemia-reperfusion.

FIG. 10 shows the inhibition of arthritic right knee joint swelling byAcF-[OPdChaWR] given orally on Days 2 to +14.

FIG. 11 shows the inhibition of right knee joint TNF-α and IL-6 levelsin joint lavage. “Untreated” refers to animals not treated withAcF-[OPdChaWR] but with the right knee challenged with antigen followingsensitisation.

FIG. 12 shows that dermal application of 3D53 in DMSO/distilled H₂O orPG/H₂O results in the appearance of the C5a antagonist in thecirculating plasma within 15 minutes, and that significant levelspersist for at least four hours. Points represent the mean±SEM in eachgroup (n=6-8).

FIG. 13 shows the inhibition of C5a-induced neutropenia by topicaladministration of C5a antagonists. The results are expressed aspercentage change from a zero time baseline.

FIG. 14A, FIG. 14B, and FIG. 14C that topical administration of C5aantagonists inhibits systemic effects of intravenously administered LPSin rats. The data show that administration of various C5a antagonists,either i.v. (1 mg/kg), or topically by dermal application (50 mg/kgtotal applied dose: solvent vehicle 50% DMSO/50% H₂O), inhibits theneutropenia caused by i.v. LPS (1 mg/kg). (FIG. 14A) 3D53 (compound 1);(FIG. 14B) compound 45; (FIG. 14C) compound 17.

FIG. 15A and FIG. 15B show the effects of the C5a antagonistAcF-[OPdChaWR] on increases in serum levels of (FIG. 15A) creatinekinase (CK) and (FIG. 15B) lactate dehydrogenase (LDH) duringreperfusion in rats. Data represent the mean±SEM (n=6-10). *P<0.05 alldrug-treated groups vs. ischemia/reperfusion (I/R)-only; † P<0.05 alldrug-treated groups vs. sham-operated.

FIG. 16A and FIG. 16B show the effects of the C5a antagonist,AcF-[OPdChaWR], on increases in serum levels of (FIG. 16A) alanineaminotransferase (ALT) and (FIG. 16B) aspartate aminotransferase (AST)following 2, 3 and 4 hours reperfusion in rats. Data represent themean±SEM (n=6-10). * P<0.05 all drug-treated groups vsischemia/reperfusion (I/R)-only; † P<0.05 all drug-treated groups vs.sham-operated.

FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D show the levels of (FIG. 17A)circulating PMNs, (FIG. 17B) muscle myeloperoxidase (MPO), (FIG. 17C)lung MPO and (FIG. 17D) liver MPO in rats. Levels were measured in ratsat the completion of the experiment. Data represent the mean±SEM(n=4-10). * P<0.05 vs. ischemia/reperfusion (I/R)-only; † P<0.05 vs.sham-operated.

FIG. 18 shows the levels of tumour necrosis factor-α (TNF-α) in ratliver homogenate samples taken at the completion of the experiment. Datarepresent the mean±SEM (n=4-10). * P<0.05 vs. ischemia/reperfusion(I/R)-only; † P<0.05 vs. sham-operated.

FIG. 19 shows the amount of edema (wet-to-dry ratio) in the hindlimbmuscle of rats at the completion of the experiment. Data represent themean±SEM (n=4-10). * P<0.05 vs. ischemia/reperfusion (I/R)-only; †P<0.05 vs. sham-operated.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsign or symptom thereof, and/or may be therapeutic in terms of a partialor complete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a vertebrate, a mammal,particularly a human, and includes: preventing the disease fromoccurring in a subject who may be predisposed to the disease, but hasnot yet been diagnosed as having it; inhibiting the disease, i.e.,arresting its development; or relieving or ameliorating the effects ofthe disease, i.e., cause regression of the effects of the disease.

The invention includes various pharmaceutical compositions useful forameliorating disease. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing a compound offormula I, analogues, derivatives or salts thereof and one or morepharmaceutically-active agents or combinations of compound of formula Iand one or more pharmaceutically-active agents into a form suitable foradministration to a subject using carriers, excipients and additives orauxiliaries.

Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Wilkins (2000) and The British NationalFormulary 43rd ed. (British Medical Association and Royal PharmaceuticalSociety of Great Britain, 2002), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dosage units. Solid dosage units include tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orby administration of several smaller dose units, and also by multipleadministrations of subdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration or the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the cytotoxic side effects. Variousconsiderations are described, e.g., Langer (Science, 249:1527, 1990).Formulations for oral use may be in the form of hard gelatin capsules,in which the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin. They may alsobe in the form of soft gelatin capsules, in which the active ingredientis mixed with water or an oil medium, such as peanut oil, liquidparaffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may be suspending agents such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents, which may be (a) a naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkylenoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents such as those mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1, 3-butanediol. Among the acceptablevehicles and solvents which may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed, includingsynthetic mono- or triglycerides. In addition, fatty acids such as oleicacid may be used in the preparation of injectables.

Compounds of formula I may also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

Dosage levels of the compound of formula I of the present invention willusually be of the order of about 0.5 mg to about 20 mg per kilogram bodyweight, with a preferred dosage range between about 0.5 mg to about 10mg per kilogram body weight per day (from about 0.5 g to about 3 g perpatient per day). The amount of active ingredient which may be combinedwith the carrier materials to produce a single dosage will vary,depending upon the host to be treated and the particular mode ofadministration. For example, a formulation intended for oraladministration to humans may contain about 5 mg to 1 g of an activecompound with an appropriate and convenient amount of carrier material,which may vary from about 5 to 95 percent of the total composition.Dosage unit forms will generally contain between from about 5 mg to 500mg of active ingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

In addition, some of the compounds of the invention may form solvateswith water or common organic solvents. Such solvates are encompassedwithin the scope of the invention.

The compounds of the invention may additionally be combined with othercompounds to provide an operative combination. It is intended to includeany chemically compatible combination of pharmaceutically-active agents,of the compound of formula I of this invention.

It will be clearly understood that the foregoing comments regardingpharmaceutical formulations, routes of administration, dosage levels andthe like are equally applicable to compound 1.

Abbreviations used herein are as follows:

-   -   BOP benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium        hexafluorophosphate    -   C5cR C5a receptor    -   dH₂O distilled water    -   D-Cha D-cyclohexylamine    -   DIPEA diisopropylethylamine    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulphoxide    -   HBTU O-benzotriazole N′,N′,N′,N′-tetramethyluronium        hexafluorophosphate    -   HEPES N-[2-hydroxyethyl]piperazine-N′-[2-ethane sulfonic acid]    -   HPLC high performance liquid chromatography    -   RP-HPLC reverse phase high performance liquid chromatography    -   i.v. intravenous    -   LPS lipopolysaccharide    -   PMN polymorphonuclear granulocyte    -   p.o. oral    -   RMSD root mean square deviation    -   rp-HPLC reverse phase-high performance liquid chromatography    -   TFA trifluoroacetic acid;

Throughout the specification conventional single-letter and three-lettercodes are used to represent amino acids.

The terms “3D53” and “PMX53” are synonymous, and represent the compoundAc-Phe-[Orn-Pro-dCha-Trp-Arg];

The terms “LP-10” and “PMX-201” are synonymous, and represent thecompound Ac-Phe-[Orn-Pro-dCHa-Trp-Cit]; and

The terms “LP-16” and “PMX-205” are synonymous, and represent thecompound EC—HC—[Orn-Pro-dCha-Trp-Arg], in which “HC” indicateshydrocinnamate.

The invention will now be described by way of reference only to thefollowing general methods and experimental examples.

We have found that all of the compounds of formula I which have so farbeen tested have broadly similar pharmacological activities, which aresimilar to those of compound 1 (also referred to herein as 3D53 orPMX53), although the physicochemical properties, potency, andbioavailability of the individual compounds varies somewhat depending onthe specific substituents. Thus we expect that results obtained in vitroor in vivo with compound 1 will be reasonably predictive of activity ofthe compounds of formula I in corresponding assays.

General Methods

Protected amino acids and resins were obtained from Novabiochem. TFA,DIPEA and DMF (peptide synthesis grade) were purchased from Auspep. Allother materials were reagent grade unless otherwise stated. Preparativescale reverse-phase HPLC separations were performed on a Vydac C18reverse-phase column (2.2×25 cm), and analytical reverse-phase HPLCseparations were performed on a Waters Delta-Pak PrepPak C18reverse-phase column (0.8×10 cm), using gradient mixtures of solventA=water/0.1% TFA and solvent B=water 10%/acetonitrile 90%, 0.09% TFA.The molecular weight of the peptides was determined by electrospray massspectrometry, recorded on a triple quadruple mass spectrometer (PE SCIEXAPI III), as described elsewhere (Haviland et al, 1995). ¹H-NMR spectrawere recorded on either a Bruker ARX 500 MHz or a Varian Unity 400spectrometer. Proton assignments were determined by 2D NMR experiments(DFCOSY, TOCSY, NOESY). Compounds were analysed by mass spectrometry andby reversed phase analytical HPLC.

Compound Synthesis

Linear peptide sequences were assembled by manual step-wise standardsolid-phase peptide synthesis (SPPS) techniques well known to thoseskilled in the art. The amino acids or peptide termini were activatedwith HBTU with DIEA in situ neutralisation. Couplings were monitored bythe standard quantitative ninhydrin test. Boc chemistry was employed fortemporary Nα-protection of amino acids with two 1 minute treatments withTFA for Boc group removal. Peptides were synthesised on a NovabiochemBoc-D-Arg(Tos)-PAM or Boc-L-Arg(Tos)-PAM resin with a substitution valueof approx. 0.2-0.5 mmol/g. The peptides were fully deprotected andcleaved by treatment with liquid HF (10 mL), p-cresol (1 mL) at −5° C.for 1-2 hrs. Peptides were purified by reversed phase HPLC (e.g.,gradient: 0% B to 75% B over 60 min) and analysed by electrospray massspectrometry.

Alternatively, the linear peptides can be synthesised by Fmoc chemistry,using HBTU/DIEA activation on an Fmoc-D-Arg (Mtr)-Wang resin. Fmoc groupremoval was effected using two 1 min treatments with 50% piperidine/DMF.Cleavage and deprotection using 95% TFA/2.5% TIPS/2.5% H2O gives theMtr-protected peptide, which can be purified by RP-HPLC. A generalprocedure for cyclization of linear peptides involves dissolving thepeptide (1 equiv.) and BOP (5 equiv.) in DMF (10 mM peptideconcentration) and stirring vigorously, followed by the addition of DIEA(15 equiv.). Solutions are generally allowed to stir at room temperatureovernight, although in most cases the reaction was complete within 2hrs. DMF is removed under high vacuum at 30° C. on a rotary evaporatorand then purified by RP-HPLC. For cyclic peptides containing a freeN-terminus, an Fmoc group was used as the temporary N-terminalprotecting group during the cyclization step. DMF was removed under highvacuum at 30° C. on a rotary evaporator, and then the peptide wastreated with 30% piperidine/DMF for 1 hr at room temperature to removethe Fmoc group. This was followed by solvent removal under high vacuum,and purification by RP-HPLC. Representative examples of the synthesis ofthe cycles are described below.

NMR Structure Determination

¹H-NMR spectra were recorded for test compounds (3 mg in 750 μL,d₆-DMSO, δ 2.50) referenced to solvent on a Varian Unity 400spectrometer at 24° C. Two-dimensional ¹H-NMR NOESY (relaxation delay2.0 s, mix time 50-300 ms), DFQ-COSY and TOCSY (mixing time 75 ms)experiments were acquired and recorded in phase sensitive mode.Acquisition times=0.186 s, spectral width=5500 Hz, number of complexpoints (t₁ dimension)=1024 for all experiments. Data was zero-filled andFourier transformed to 1024 real points in both dimensions.

NMR data for compound 1 was processed using TRIAD software (TriposAssoc.) on a Silicon Graphics Indy work station. 2D NOE cross peaks wereintegrated and characterised into strong (1.8-2.5 Å), medium (2.3-3.5 Å)and weak (3.3-5.0 Å). Preliminary three-dimensional structures werecalculated from upper and lower distance limit files using Diana 2.8 (69distance constraints, including 27 for adjacent residues and 6 furtheraway) with the redundant dihedral angle constraints (REDAC) strategy.Upper and lower distance constraints were accurately calculated usingMARDIGRAS. At this stage the peptide was examined for possible hydrogenbonds, and these were added as distance constraints. The 50 lowestenergy Diana structures were subjected to restrained molecular dynamics(RMD) and energy minimisation (REM). Initially, REM consisted of a 50step steepest descent followed by 100 step conjugate gradientminimisation. RMD was performed by simulated heating of the structuresto 300° K for 1 ps, followed by 500° K for 1 ps. The temperature wasgradually lowered to 300° K over 2 ps and finally for 2 ps at 200° K.REM was performed again with a 50 step steepest descent, 200 stepconjugate gradient followed by a 300 step Powell minimisation. The finalstructures were examined to obtain a mean pairwise rms difference overthe backbone heavy atoms (N, Cα and C). Twenty of the 50 structures hada mean rmsd<Å for all backbone atoms (O, N, C).

Receptor-Binding Assay

Assays were performed with fresh human PMNs, isolated as previouslydescribed (Sanderson et al, 1995), using a buffer of 50 mM HEPES, 1 mMCaCl₂, 5 mM MgCl₂, 0.5% bovine serum albumin, 0.1% bacitracin and 100 μMphenylmethylsulfonyl fluoride (PMSF). In assays performed at 4° C.,buffer, unlabelled human recombinant C5a (Sigma) or peptide,Hunter/Bolton labelled ¹²⁵I-C5a (˜20 pM) (New England Nuclear, MA) andPMNs (0.2×10⁶) were added sequentially to a Millipore Multiscreen assayplate (HV 0.45) having a final volume of 200 μL/well. After incubationfor 60 min at 4° C., the samples were filtered and the plate washed oncewith buffer. Filters were dried, punched and counted in an LKB gammacounter. Non-specific binding was assessed by the inclusion of 1 mMpeptide or 100 nM C5a, which typically resulted in 10-15% total binding.

Data was analysed using non-linear regression and statistics withDunnett post-test.

Myeloperoxidase Release Assay for Antagonist Activity

Cells were isolated as previously described (Sanderson et al, 1995) andincubated with cytochalasin B (5 μg/mL, 15 min, 37° C.). Hank's BalancedSalt solution containing 15% gelatin and peptide was added on to a 96well plate (total volume 100 μL/well), followed by 25 μL cells(4×106/mL). To assess the capacity of each peptide to antagonise C5a,cells were incubated for 5 min at 37° C. with each peptide, followed byaddition of C5a (100 nM) and further incubation for 5 min. Then 50 μL ofsodium phosphate (0IM 0.1M, pH 6.8) was added to each well, the platewas cooled to room temperature, and 25 μL of a fresh mixture of equalvolumes of dimethoxybenzidine (5.7 mg/mL) and H2O2 (0.51%) was added toeach well. The reaction was stopped at 10 min by addition of 2% sodiumazide. Absorbances were measured at 450 nm in a Bioscan 450 platereader, corrected for control values (no peptide), and analysed bynon-linear regression.

In vivo Assays of Anti-Inflammatory Activity

The following well-known in vivo assay systems are used to assess theanti-inflammatory activity of compounds of the invention. All assay dataare analysed using non-linear regression analysis and Student's t-test,analysis of variance, with p<0.05 as the threshold level ofsignificance.

(a) Carrageenan Paw Oedema

Anaesthetised (i.p. ketamine & xylazine) Wistar rats (150-200 g) or miceare injected with sterilised air (20 mL day 1, 10 mL day 4) into thesubcutaneous tissue of the back. The cavity can be used after 6 days,whereupon carrageenan (2 mL, 1% w/w in 0.9% saline) is injected into theair pouch, and exudate is collected after 10 hr. Test compounds areadministered daily after Day 6, and their anti-inflammatory effectsassayed by differential counting of cells in the air-pouch exudate.Animals are killed at appropriate times after injection, and 2 mL 0.9%saline is used to lavage the cavity; lavage fluids are transferred toheparinised tube and cells are counted with a haemocytometer andDiff-Quik stained cytocentrifuged preparation.

Alternatively, a routine carrageenan paw oedema developed in Wistar ratsby administering a pedal injection of carrageenan may be used to elicitoedema which is visible in 2 h and maximised in 4 h. Test compounds aregiven 40 min before inflammagen and evaluated by microcalipermeasurements of paws after 2 & 4 hr. See Fairlie, D. P. et al 1987).Also see Walker and Whitehouse (1978).

(b) Adjuvant Arthritis

Adjuvant arthritis is induced in rats (3 strains) either immunologically(injection of heat-killed Mycobacterium tuberculosis) or chemically(with pyridine) by inoculation with the arthritogenic adjuvantco-administered with oily vehicles, such as Freund's adjuvants in thetail base (See Whitehouse, M. W., Handbook of Animal Models for theRheumatic Diseases, Eds. Greenwald, R. A.; Diamond, H. S.; Vol 1, pp.3-16, CRC Press).

Within 13 days the adjuvant arthritis is manifested by localinflammation and ulceration in the tail, gross swelling of all fourpaws, inflammatory lesions in paws and ears, weight loss and fever.These symptoms, which are similar to those of inflammatory disease inhumans (Winter and Nuss, 1966), can be alleviated by agents such asindomethacin or cyclosporin, which also show beneficial effects in man(e.g., Ward and Cloud, 1966). Without drug treatment at Day 14,arthritic rats had hypertrophy of the paws, reduced albumin but raisedacute phase reaction proteins in serum, and depressed hepatic metabolismof xenobiotics as indicated by prolonged barbiturate-induced sleepingtimes.

To assess activity, compounds are administered for 4 days orally (<10mg/kg/day) or intraperitoneally (i.p.) from Days 10-13 followinginoculation with arthritogen (Day 0). If the compound is active, theinflammation is either not visible, or is very significantly reduced inrear or front paws, as assessed by microcaliper measurements of pawthickness and tail volume, as well as by gross inspection ofinflammatory lesions. Animals are sacrificed by cervical dislocation onDay 18 unless arthritis signs are absent, whereupon duration ofobservations is continued with special permission from the EthicsCommittees. Experiments are staggered to maximise throughput and allowearly comparisons between compounds. This routine assay is well-acceptedas identifying anti-inflammatory agents for use in humans.

Pharmacokinetics

Female and male Wistar rats (200-250 g) were anaesthetized with 1 mL ofzoletil (50 mg/kg) and xylazine (10 mg/kg; Lyppard, Australia), whichwas injected intraperitoneally. An area of 5×10 cm was shaved and markedon the lower abdominal area of the rat, on to which the dose of drug wasapplied. A stock solution containing 10 mg/ml of C5a antagonist wasdissolved in solvents propylene glycol or dimethylsulfoxide at varyingconcentrations with water and smeared evenly on the shaved abdominalarea of the rat with a spatula. A heating pad was used to maintain thebody temperature of the rats and blood samples were taken at 15 minintervals for the first hour, and after that at 1 hr intervals for atotal period of 3 hours.

Blood samples were immediately added to tubes containing heparin (500Units/μL) and centrifuged (11000×g). The plasma layer of each sample wasremoved and stored at −20° C. A deuterated internal standard,²H3CO—F-[OPdChaWR] 50 μL, 5 μg/mL in 50% aeotonitrile/water), was addedand vortexed. The samples were further diluted 1:3 with high performanceliquid chromatography (HPLC) grade acetonitrile and rapidly vortexed (20sec), then centrifuged (11000×g). This process resulted in precipitationof large plasma proteins in the samples, and allowed the completeextraction of the drug from the plasma. The fluid portions of thesamples were placed in 1 mL Eppendorf tubes and stored until analysed.

These samples were transferred to 96-well plates and evaporated todryness using a GeneVac centrifugal evaporator, then reconstituted inthe wells with mobile phase (20 μL). Analysis of samples was performedby liquid chromatography (LCMS) using an Agilent 1100 series HPLCequipped with a well plate autosampler coupled with a PE Sciex QstarPulsar ESI-TOF mass spectrometer. Concentrations were determined from astandard curve of drug: internal standard peak area ratios. Standardswere prepared by adding an appropriate amount of the drug and internalstandard to plasma from an untreated rat, and were extracted andprepared by the same method as the experimental samples.

Example 1 Synthesis of Cyclic Compounds

Synthesis of cycle AcF-[OPdChaWR] (1). The linear peptideAc-Phe-Orn-Pro-dCha-Trp-Arg was synthesised by Boc chemistry on a 0.20mmole scale using HBTU/DIEA activation and in situ neutralization on aBoc-L-Arg(Tos)-PAM resin (338 mg, SV=0.591 mmol/g). Cleavage anddeprotection of the resin (457 mg) was achieved by treating the resinwith HF (10 mL) and p-cresol (1 mL) at −5 to 0° C. for 1-2 hrs, to givecrude peptide (160 mg, 90%). Cyclization involved stirring the crudepeptide (41 mg, 45 μmol), BOP (126 mg, 0.28 mmol) and DIEA (158 μL, 0.9mmol) in DMF (57 mL) for 15 hrs. The solvent was removed in vacuo andthe cyclic peptide purified by rpHPLC (18.8 mg, 47%) Rt=10.8 min(gradient: 70% A/30% B to 0% A/100% B over 30 min). MS:[M+H]+(calc.)=896.5, [M+H]+(exper.)=896.5.

Synthesis of cycle AcF-[OPdPheWR] (33). The linear peptideAc-Phe-Orn-Pro-dPhe-Trp-Arg was synthesised by Boc chemistry usignHBTU/DIEA activation and in situ neutralisation on a Boc-L-Arg (Tos)-PAMresin. Cleavage and deprotection of the resin was achieved by treatingthe resin with HF (10 mL) and p-cresol (1 mL) at −5 to 0° C. for 1-2hrs, to give crude peptide. Cyclization involved stirring the crudepeptide (85 mg), BOP (200 mg) and DIEA (222 μL) in DMF (10 mL) for 15hrs. The solvent was removed in vacuo and the cyclic peptide purified byrpHPLC (31 mg) Rt=16. 7 min (gradient: 70% A/30% B to 0% A/100% B over30 min). MS: [M+H]+(calc.)=890.5, [M+H]+(exper.)=890.5.

Synthesis of cycle AcF-[OPdChaFR] (60). The linear peptideAc-Phe-Orn-Pro-dCha-Phe-Arg was synthesised by Boc chemistry usingHBTU/DIEA activation and in situ neutralization on a Boc-L-Arg(Tos)-PAMresin. Cleavage and deprotection of the resin was achieved by treatingthe resin with HF (10 mL) and p-cresol (1 mL) at −5 to 0° C. for 1-2hrs, to give crude peptide. Cyclization involved stirring the crudepeptide (104 mg), BOP (57 mg) and DIEA (103 mL) in DMF (1 mL) for 15hrs. The solvent was removed in vacuo and the cyclic peptide purified byrpHPLC (52 mg). Rt=11.37 min (gradient: 70% A/30% B to 0% A/100% B over15 min). MS: [M+H]+(calc.)=857.5, [M+H]+(exper.)=857.4.

Synthesis of cycle AcF-[OpdCha(N-Me-Phe)R] (64). The linear peptideAc-Phe-Om-Pro-dCha-(N-Me-Phe)-Arg-OH was synthesised by Fmoc chemistryusing HBTU/DIEA activation and in situ neutralisation on a Fmoc-L-Arg(pbf)-Wang resin (0.35 mmol/g) from Novabiochem using methyl-L-Phe (281mg, 2 equiv), Fmoc-dCha (275 mg, 2 equiv), Fmoc-Pro (472 mg, 4 equiv),Fmoc-Orn (Boc) (477 mg, 3 equiv), Fmoc-Phe (542 mg, 4 equiv) and Ac2O (4equiv). Cleavage and deprotection of the resin was achieved by treatingthe resin with 95% TFA (15 mL) for 1 h to give crude peptide (150 mg)after precipitation with diethyl ether. Cyclization involved stirringthe rpHPLC purified peptide (100 mg), BOP (200 mg) and DIEA (222 μL) inDMF (2 mL) for 4 hrs. The solvent was removed in vacuo and the cyclicpeptide purified by rpHPLC (50 mg) Rt=33 min (gradient: 70% A/30% B to0% A/100% B over 30 min). MS: [M+H]+(calc.)=871.5, [M+H] (exper.)=871.5.

Synthesis of cycle AcF-[{Orn-(δN-Me)}PdChaWR] (66). Boc-(δN-Me-Orn)-OHwas synthesized as reported (Pol. J. Chem. 1988, 62, 257-261). Thelinear peptide Ac-Phe-[Orn-(δN-MeCbz)]-Pro-dCha-Trp-arg-OH wassynthesised by Boc chemistry using HBTU/DIEA activation and in situneutralisation on Boc-L-Arg(tosyl)PAM resin (0.41 mmol/g) fromNovabiochem. Cleavage and deprotection of the resin was achieved bytreating the resin with HF/pCresol for 2 h to give crude peptide afterprecipitation with diethyl ether. Cyclization involved stirring theRP-HPLC purified peptide (100 mg), BOP (200 mg) and DIEA (222 μL) in DMF(2 mL) for 4 hrs. The solvent was removed in vacuo and the cyclicpeptide purified by rpHPLC. Rt=11.5 min (35% B). MS:[M+H]+(calc.)=910.5, [M+H]+(exper.)=910.5.

Purification and Characterization.

Crude peptides were purified using preparative rp-HPLC using a Vydac C18reverse-phase column (2.2×25 cm). Gradients of 1 mL/min of solvent A tosolvent B were employed and monitored at 214 nm. Fractions werecollected and tested by ion spray mass spectrometry (ISMS) for thecorrect molecular weight, and purity was checked by analytical rp-HPLCon a Waters Delta-Pak PrepPak® C18 reverse-phase column (0.8×10 cm)(varying gradients such as: 0 to 75% over 60 min). The acetonitrile wasHPLC grade (BDH Laboratories) and TFA was synthesis grade (Auspep).

Table 1 shows examples of reactions used to prepare cyclic compounds1-70, and their characterisation by electrospray mass specrometry (MassSpec Found) and reversed phase HPLC (rp-HPLC) retention times (Rt mins)under specified elution conditions.

Table 2 depicts the structures of the respective compounds, and liststheir respective receptor binding affinities and antagonist potenciesfor the C5a receptor on human polymorphonuclear leukocytes(neutrophils), as measured by myeloperoxidase assay.

TABLE 1 Summary of Synthesis and Characterisation of Cyclic Compoundslisted in Table 2 Amount Mass Mass Linear Amount Amount Amount YieldSpec Spec rpHPLC Compound Peptide BOP DIPEA DMF Cycle Calcd Found rpHPLCconditions Rt (min) 1 41 mg 126 mg  158 μL 57 mL  19 mg 895.5 896.530→100% B 30 m 10.8 2 957.5 958.5 30→100% B 30 m 14.8 3 78 mg 36 mg 71μL 1 mL 29 mg 937.6 938.5 30→100% B 30 m 13.1 4 65 mg 29 mg 57 μL 1 mL966.6 967.5 30→100% B 60 m 18.5-20.5 5 81 mg 36 mg 71 μL 1 mL 967.5968.5 30→100% B 90 m 23.2 6 25 mg — 0.25 mL 2M 0.5 mL Quantitative Asabove 19.3 NaOH MeOH 7 92 mg 200 mg  222 μL 10 mL  29 mg 910.5 910.330→100% B 30 m 23.0-23.8 8 87 mg 200 mg  222 μL 10 mL  18 mg 896.5 898.430→100% B 30 m 13.1-14.2 9 155 mg  200 mg  222 μL 10 mL   5 mg 912.5912.5 30→100% B 30 m 3.2-4.2 10 105 mg  200 mg  222 μL 10 mL  51 mg932.5 932.7 30→100% B 30 m 6.0-8.2 11 88 mg 200 mg  222 μL 10 mL  27 mg1008.5 1008.7 30→100% B 30 m 10.4-11.7 12 72 mg 200 mg  222 μL 10 mL  15mg 882.5 882.5 30→100% B 30 m 7.0-8.0 13 53 mg 28 mg 56 μL 1 mL 21 mg805.5 806.5 30→100% B 60 m 7.6 14 51 mg 200 mg  222 μL 10 mL  12 mg914.5 914.5 30→100% B 30 m 8.6-9.4 15 90 mg 200 mg  222 μL 10 mL  33 mg914.5 914.5 30→100% B 30 m 7.3-8.5 16 81 mg 200 mg  222 μL 10 mL  22 mg935.5 935.5 30→100% B 30 m 21.8-22.2 17 90 mg 46 mg 91 μL 1 mL 46 mg838.5 839.4 30→100% B 60 m 13.4-17   18 60 mg 31 mg 61 μL 1 mL 25 mg836.5 837.4 30→100% B 60 m 15.1-18   19 53 mg 33 mg 65 μL 1 mL 691.4692.4 30→100% B 60 m   9-10.5 20 66 mg 200 mg  222 μL 10 mL  15 mg 898.5898.5 30→100% B 30 m 14.2-14.7 21 81 mg 200 mg  222 μL 10 mL  22 mg912.5 912.5 30→100% B 30 m 17.2-18.0 22 59 mg 200 mg  222 μL 10 mL  20mg 945.5 946.7 30→100% B 30 m 24 1110 mg  200 mg  222 μL 10 mL  37 mg952.6 952.4 30→100% B 30 m 16.7-17.2 25 82 mg 200 mg  222 μL 10 mL  20mg 912.5 912.5 30→100% B 30 m 7.2-8.3 26 71 mg 200 mg  222 μL 10 mL  16mg 928.5 928.5 30→100% B 30 m 6.7-7.6 27 75 mg 200 mg  222 μL 10 mL  21mg 896.5 896.7 30→100% B 30 m 25.9-26.3 28 130 mg  62 mg 122 μL 2 mL 90mg 909.5 910.4 30→100% B 60 m 14.6-17.6 29 143 mg  73 mg 144 μL 2 mL 60mg 841.4 842.5 30→100% B 60 m 7.4-8.9 30 135 mg  72 mg 142 μL 2 mL 813.4814.4 30→100% B 60 m 16.8-18.6 31 102 mg  52 mg 101 μL 2 mL 855.5 856.630→100% B 60 m 8.2-9.7 32 49 mg 200 mg  222 μL 1 mL  8 mg 928.5 929.630→100% B 30 m 12.7-13.7 33 85 mg 200 mg  222 μL 10 mL  31 mg 889.5890.5 30→100% B 30 m 16.5-16.9 34 122 mg  57 mg 113 μL 10 mL  901.5902.2 30→100% B 15 m 6.0 35 106 mg  49 mg 95 μL 2 mL 901.5 902.2 30→100%B 15 m 10.7 36 120 mg  61 mg 122 μL 2 mL 855.5 856.4 30→100% B 60 m 9.2-10.7 39 70 mg 200 mg  222 μL 2 mL 28 mg 906.5 906.7 30→100% B 60 m40 62 mg 34 mg 66 μL 10 mL 799.4 800.6 30→100% B 60 m   9-9.7 44 100 mg 48 mg 93 μL 1 mL 909.5 910.6 30→100% B 60 m 13.7-16.3 45 94 mg 45 mg 89μL 1 mL 896.5 897.8 30→100% B 60 m 14.0-15.2 49 55 mg 29 mg 58 μL 1 mL796.4 797.4 30→100% B 60 m 17.2-18.6 50 75 mg 30 mg 0.06 mL 1 mL 35 mg862.5 863.7 30→100% B 90 m 21-23 51 64 mg 27 mg 0.05 mL 1 mL 25 mg 822.5823.7 30→100% B 90 m 14.5-17   52 169 mg  94 mg 184 μL 1 mL 67 mg 906.5907.5 30→100% B 60 m   17-18.4 53 177 mg  84 mg 166 μL 2 mL 906.5 907.630→100% B 60 m 16.1-20.4 54 79 mg 200 mg  222 μL 2 mL 22 mg 944.5 945_530→100% B 30 m 56 161 mg  79 mg 156 μL 10 mL  868.5 869.2 30→100% B 60 m57 70 mg 39 mg 70 μL 2 mL 846.5 847.4 30→100% B 60 m 15.3-17.6 58 160mg  76 mg 150 μL 1 mL 912.5 913.3 30→100% B 60 m 15.4-19.6 59 150 mg  73mg 143 μL 2 mL 895.5 896.6 30→100% B 15 m 10.4 60 856.5 857.4 30→100% B15 m 11.4 61 160 mg  80 mg 156 μL 2 mL 870.5 871.4 30→100% B 60 m13.9-17.4 62 180 mg  91 mg 180 μL 2 mL 84 mg 850.4 851.4 30→100% B 60 m 8.9-11.3 63 174 mg  83 mg 164 μL 2 mL 75 mg 900.5 901.4 30→100% B 60 m13.4-15.3 64 100 mg  200 mg  222 μL 2 mL 50 mg 870.5 871.5 30→100% B 60m 33.0 65 100 mg  200 mg  222 μL 2 mL 903.5 904.5 35% B 8.2 66 100 mg 200 mg  222 μL 2 mL 909.5 910.5 35% B 11.5 67 50 mg 50 mg 100 μL 5 mL 22mg 861.6 862.6 30→100% B 30 m 20.5 68 50 mg 50 mg 100 μL 5 mL 18 mg881.5 882.4 30→100% B 30 m 29.5 69 50 mg 50 mg 100 μL 5 mL 19 mg 863.0864.0 30→100% B 30 m 23.2 70 50 mg 50 mg 100 μL 5 mL  4 mg* 884.0 885.030→100% B 30 m 29.5 *Tyr-O-Benzyl was the amino acid used, but theproduct involved a rearrangement to a meta-C-substituted tyrosine with abenzyl substituent (see structure of compound 70).

Example 2 Antagonist Activity of Cyclic Compounds

Table 2 shows the structures of the compounds synthesised in Example 1,as well as their respective receptor binding affinities and antagonistpotencies for the C5a receptor on human polymorphonuclear leukocytes(neutrophils), as measured by the myeloperoxidase assay.

Compounds 1-9, 16-18, 20, 21, 23, 24, 27-32, 36, 38, 44, 51, and 59 arewithin the broad scope of the general structure set out in our earlierpatent application, Intl. Pat. Appl. No. PCT/AU98/00490. However, Table2 demonstrates that in fact, of these compounds only 1-6, 17, 20, 28,30, 31, 36 and 44 have appreciable antagonist potency (IC₅₀<1 μM)against the C5a receptor on human neutrophils. The other compounds, 7-9,16, 18, 21, 23, 24, 27, 29, 32, 38, 51, and 59, do not show appreciableantagonist potency and/or receptor affinity, with IC₅₀>1 μM in allcases.

On the other hand, compounds 10-15, 19, 22, 25, 26, 33-35, 37, 39-43,45, 47-50, 52-58, and 60-70 are not included within the scope of Intl.Pat. Appl. No. PCT/AU98/00490, although they do involve the same orsimilar cyclic scaffolds to those disclosed therein. Table 2 shows thatof these new compounds, 10-12, 14, 15, 25, 33, 35, 40, 45, 48, 52, 58,60, 66 and 68-70 have appreciable antagonist potency (IC₅₀<1 μM).However, the other compounds (13, 19, 22, 26, 34, 37, 39, 41-43, 47, 49,50, 53-57, 61-65, and 67) do not show appreciable antagonist potencyand/or receptor affinity, with >1 μM in all these cases.

The results shown in Table 2 enable us to define further and to refinethe limitations on the active pharmacophore for C5a receptor antagonistactivity, in order to obtain or predict sub-micromolar antagonistpotency.

TABLE 2 Strμctμres and Activities of 70 Examples of Cyclic Antagonistsof C5a Receptors on Hμman Polymorphonμclear Leμkocytes

1 C5aR Binding IC₅₀: 0.45 μM C5aR Antagonist IC₅₀: 28 nM

2 C5aR Binding IC₅₀: 1.1 μM C5aR Antagonist IC₅₀: 110 nM

3 C5aR Binding IC₅₀: 0.84 μM C5aR Antagonist IC₅₀: 0:30 nM

4 C5aR Binding IC₅₀: 0.25 μM C5aR Antagonist IC₅₀: 62 nM

5 C5aR Binding IC₅₀: 0.84 μM C5aR Antagonist IC₅₀: 38 nM

6 C5aR Binding IC₅₀: 0.45 μM C5aR Antagonist IC₅₀: 23 nM

7 C5aR Binding IC₅₀: 1000 μM C5aR Antagonist IC₅₀: ND

8 C5aR Binding IC₅₀: 28.7 μM C5aR Antagonist IC₅₀: ND

9 C5aR Binding IC₅₀: 0.** μM C5aR Antagonist IC₅₀: ND

10 C5aR Binding IC₅₀: 0.47 μM C5aR Antagonist IC₅₀: 34 nM

11 C5aR Binding IC₅₀: 0.96 μM C5aR Antagonist IC₅₀: 291 nM

12 C5aR Binding IC₅₀: 0.76 μM C5aR Antagonist IC₅₀: 151 nM

13 C5aR Binding IC₅₀: 37 μM C5aR Antagonist IC₅₀: ND

14 C5aR Binding IC₅₀: 0.52 μM C5aR Antagonist IC₅₀: 38 nM

15 C5aR Binding IC₅₀: 0.39 μM C5aR Antagonist IC₅₀: ND

16 C5aR Binding IC₅₀: 19.2 μM C5aR Antagonist IC₅₀: ND

17 C5aR Binding IC₅₀: 0.22 μM C5aR Antagonist IC₅₀: 31 nM

18 C5aR Binding IC₅₀: 9.9 μM C5aR Antagonist IC₅₀: ND

19 C5aR Binding IC₅₀: 16.1 μM C5aR Antagonist IC₅₀: ND

20 C5aR Binding IC₅₀: 0.68 μM C5aR Antagonist IC₅₀: ND

21 C5aR Binding IC₅₀: 2.9 μM C5aR Antagonist IC₅₀: ND

22 C5aR Binding IC₅₀: 2.4 μM C5aR Antagonist IC₅₀: ND

23 C5aR Binding IC₅₀: 2.4 μM C5aR Antagonist IC₅₀: ND

24 C5aR Binding IC₅₀: >1000 μM C5aR Antagonist IC₅₀: ND

25 C5aR Binding IC₅₀: 0.27 μM C5aR Antagonist IC₅₀: ND

26 C5aR Binding IC₅₀: 75.5 μM C5aR Antagonist IC₅₀: ND

27 C5aR Binding IC₅₀: 144 μM C5aR Antagonist IC₅₀: ND

28 C5aR Binding IC₅₀: 0.39 μM C5aR Antagonist IC₅₀: 40 nM

29 C5aR Binding IC₅₀: 13 μM C5aR Antagonist IC₅₀: ND

30 C5aR Binding IC₅₀: 145 μM C5aR Antagonist IC₅₀: 37 nM

31 C5aR Binding IC₅₀: 1.1 μM C5aR Antagonist IC₅₀: 35 nM

32 C5aR Binding IC₅₀: 30.1 μM C5aR Antagonist IC₅₀: ND

33 C5aR Binding IC₅₀: 11.26 μM C5aR Antagonist IC₅₀: 22 nM

34 C5aR Binding IC₅₀: 32.1 μM C5aR Antagonist IC₅₀: ND

35 C5aR Binding IC₅₀: 9.2 μM C5aR Antagonist IC₅₀: 25 nM

36 C5aR Binding IC₅₀: 0.53 μM C5aR Antagonist IC₅₀: 30 nM

37 C5aR Binding IC₅₀: 77 μM C5aR Antagonist IC₅₀: 77 nM

38 C5aR Binding IC₅₀: 77 μM C5aR Antagonist IC₅₀: 77 nM

39 C5aR Binding IC₅₀: >1000 μM C5aR Antagonist IC₅₀: ND

40 C5aR Binding IC₅₀: 2.16 μM C5aR Antagonist IC₅₀: 30 nM

41 C5aR Binding IC₅₀: >100 μM C5aR Antagonist IC₅₀: ND

42 C5aR Binding IC₅₀: 1082 μM C5aR Antagonist IC₅₀: ND

43 C5aR Binding IC₅₀: 77 μM C5aR Antagonist IC₅₀: 77 nM

44 C5aR Binding 10₅₀: 1.36 μM C5aR Antagonist IC₅₀: 160 nM

45 C5aR Binding IC₅₀: 6.0 μM C5aR Antagonist IC₅₀: 690 nM

56 C5aR Binding IC₅₀: 4.0 μM C5aR Antagonist IC₅₀: 28 nM

57 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

58 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

59 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

60 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

61 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

62 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

63 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

64 C5aR Binding IC₅₀: 0. μM C5aR Antagonist IC₅₀: 28 nM

“C5a Binding IC₅₀” refers to the concentration of compound required toachieve 50% maximum binding to human PMNs. “C5a Antagonist IC₅₀” refersto the concentration of compound required to achieve 50% antagonism ofmyeloperoxidase release from C5a-stimulated human PMNs. Boxed regionsindicate the location of relative changes between structures. Compound 1is the lead compound from our previous application PCT/AU98/00490, andis included for purposes of comparison.

Example 3 Cyclic Antagonists of C5a

Some examples of these cyclic antagonists and their apparentreceptor-binding affinities and antagonist potencies are given in Table3, in which the single letter code for amino acids is used. “d”indicates the dexto (D) form of an amino acid. “ND” indicates notdetermined.

TABLE 3 NEW COMPOUNDS AS C5a ANTAGONISTS AcPhe Replacements Compound nBinding Antagonist MsF [OP-dCha-WR] 10 3 0.47 34 TsF [OP-dCha-WR] 11 30.96 291  AcPhg [Opd-Cha-WR] 12 3 0.76 151  AcG [OP-dCha-WR] 13 3 37.2ND Ac(o-fluoro)F[OP-dCha-WR] 14 3 0.52 38 Ac(m-fluoro)F[OP-dCha-WR] 15 10.39 ND HC [OP-dCha-WR] 17 3 0.22 31 Hydrogen [OP-dCha-WR] 19 3 >1000 NDMs = Mesyl, Ts = Tosyl, MeSuc = Methylsuccinate, Suc = Succinate, Ahx =6-Aminohexanoate, HPhe = Homophenylalanine, Phg = Phenylglycine, HC =Hydrocinnamate ND = not done Compound Binding Antagonist ProReplacements Number N (μM) (nM) AcF[O-Hyp-dCha-WR] 25 3 0.27 NDAcF[O-Thp-dCha-WR] 26 1 75 5 ND AcF[O-Phe-dCha-WR] 22 3 2.43 ND Hyp =trans-Hydroxyproline, Thp = cis-Thioproline Antagonist D-ChaReplacements Lab Code N Binding (μM) (nM) AcF[OP-dCha-WR] 3D53, 1 13 0.45 28 AcF[O-dLeu-WR] 31 3 1.13 35 AcF[OPGWR] 42 3 1082 NDAcF[OP-dVal-WR] 29 3 13.0 ND AcF[OP-dNle-WR] 36 3 0.53 30AcF[OP-dTic-WR] 35 3 9.18 15,000 AcF[OP-aic-WR] 34 3 22.71 NDAcF[OP-DTyr-WR] 40 3 2.16 300 AcF[OP-dArg-WR] 41 3 >100 NDAcF[OP-dPhe-WR] 33 3 0.26 22 AcF[OP-dhCha-WR] 28 3 0.39 40.5Aic—aminoindanecarboxylio acid Tic—tetrahydroisoquino1inedhCha—D-homocyclohexylalanine Antagonist Trp Replacements Lab Code NBinding (μM) (nM) AcF[OP-dCha-HR] 57 3 23.5 ND AcF[OP-dCha-FR] 60 3 0.2532 AcF[OP-dCha-LR] 51 3 18.9 3,000 AcF[OP-dCha-Cha-R] 50 3 11.9 4,500AcF[OP-dCha-hPhe-R] 61 3 11.5 ND AcF[OP-dCha-2Nal-R] 53 3 15.8 NDAcF[OP-dCha-Bta-R] 58 3 0.28 172 AcF[OP-dCha-Flu-R] 54 3 28.9 NDAcF[OP-dCha-1Nal-R] 52 3 0.71 46.6 AcF[OP-dCha-Tic-R] 56 3 3.73 10,900AcF[OP-dCha-G-R] 55 3 >1000 ND AcF[OPdCha-dTrp-R] 59 3 30.4 ND hPhe =Homophenylalanine, 2Nal = 2-Naphthylalanine, 1Nal = 1-Naphthylalanine,Bta = Benzothienylalanine, Flu = Fluorenylalanine, Tyr-O-alkyl =O-alkylated analogue of tyrosine. Tic = tetrahydroisoquinoline BindingAntagonist Arg Replacements Lab Code N (μM) (nM) AcF[OPdChaW-Cit] 45 36.00 690 AcF[OpdChaW-K] 47 3 24.15 ND AcF[OpdChaW-hArg] 44 3 1.36 ND Can= L-canavanine, Cit = Citrulline, hArg = homoarginine Multiple BindingAntagonist Replacements Lab Code N (μM) (nM) AcF[OP-dPhe-dleu-Nal-R] 1053 3.1 ND AcF[OP-dPhe-FR] 62 3 5.2 5,210 AcF[DapOPdChaWRC] 151 3 1.84  100 AcF[OP-dPhe-lNal-R] 63 3 3.1 ND AcF[OP-dPhe-Y-R] 150 3 69.2 ND1Nal = 1-Naphthylalanine, Dap = 2′3-diaminopropionic acid, dPhe =D-phenylalanine

Example 4 Pharmacophore Refinement

On the basis of the results in Table 2, we can develop a refinedpharmacophore for active antagonism of the C5a receptor on humanpolymorphonuclear leukocytes, as follows:

Position “A” can tolerate a very large number of groups, including H(e.g., compound 17, 18), alkyl, aryl, NH₂, NHalkyl, N(alkyl)₂, NHaryl,NHacyl (e.g., compounds 1, 3, 4, 5, 6), NHbenzoyl (e.g., compound 2),OH, Oalkyl, Oaryl, NHSO₂alkyl (e.g., compound 10), NHSO₂aryl (e.g.,compound 11), without an adverse effect on activity.

The wide tolerance to substitution at position “A” indicates that thereis considerable space in the receptor for appendages to the cyclicpeptide scaffold. This position can therefore be used for addingsubstituents in order to vary the water and lipid solubility of theantagonist, thereby enhancing oral or transdermal absorption of theantagonist. This position also allows attachment of labels such asfluorescent tags, agonists or polypeptide sequences which confer highaffinity for target cells, such as sequences similar to amino acids 1-69of C5a.

Position “B” can be alkyl, aryl, phenyl, benzyl, naphthyl or indole, orthe side chain of a D- or L-amino acid such as L-phenylalanine (compound1), or L-phenylglycine (compound 12). It should not be the side chain ofD-phenylalanine (compound 8), L-homophenylalanine (compound 7),L-tyrosine (compound 9), L-homotyrosine, glycine (compound 13),L-tryptophan (compound 16), or L-homotryptophan.

Position “B” does not tolerate a wide range of substitutents. It appearsthat the benzyl group of L-phenylalanine cannot tolerate muchsubstitution, and cannot be made much bulkier. This position seems to berequired for receptor binding, rather than being important forantagonism per se, since the greatest effects on modification were onreceptor affinity, as measured by IC₅₀.

Position “C” should be a small substitutent, such as the side chain of aD- or L-amino acid such as proline (compound 1), L-valine (compound 20),alanine, trans-hydroxyproline (compound 25), or cis-thioproline(compound 26). It should not be a bulky substituent such as the sidechains of L-isoleucine (compound 21), D- or L-phenylalanine (compounds22, 23), L-cyclohexylalanine (compound 24).

Position “D” should be a bulky substituent, such as the side chain of aD-amino acid like D-Leucine (compound 31), D-homoleucine,D-cyclohexylalanine (compound 1), D-homocyclohexylalanine (compound 28),valine (compound 29), D-norleucine (compound 36), D-homonorleucine,D-phenylalanine (compound 33), D-tetrahydroisoquinoline (compound 35),D-glutamine (compound 37), D-glutamate (compound 38), or D-tyrosine(compound 40). It should not be a smaller substituent, such as the sidechain of glycine, D-alanine (compound 30), a bulky planar side chainlike D-tryptophan (compound 32), a bulky charged side chain likeD-arginine (compound 39) or D-Lysine (compound 43), or an L-amino acidlike L-cyclohexylalanine (compound 27). Small D-amino acids and small orlarge L-amino acids at position “D” on the scaffold lead to greatlyreduced affinity for the C5a receptor.

Position “E” is chosen from among the bulky side chains of L-Tryptophan(compound 1) and L-homotryptophan, but not D-tryptophan (compound 59) orL-N-methyltryptophan (compound 47); L-phenylalanine (compound 60) butnot L-homophenylalanine (compound 61); L-naphthyl (compound 52) but notL-2-naphthyl (compound 53); L-3-benzothienylalanine (compound 58). Itshould not be the side chain of L-cyclohexylalanine (compound 50),D-leucine (compound 51), L-fluorenylalanine (compound 54), glycine(compound 55), L-tetrahydroisoquinoline (compound 56), or L-histidine(compound 57).

Substituents at position “E” on the cyclic peptide scaffold are crucialfor antagonism of the C5a receptor. Substituents at this position maylimit the conformational changes usually associated with agonistresponses. This may be a “blocking” residue, which fixes the antagonistin the receptor and prevents the conformational reorganization in thereceptor which is necessary for agonism.

Position “F” may be the side chain of L-arginine (compound 1),L-homoarginine (compound 44), L-citrulline (compound 45); orL-canavinine (compound 48). It should not be D- or L-lysine (compound47), D- or L-homolysine, or glycine (compound 49). The size of thesubstitutent at this position is important for conferring high receptoraffinity. The citrulline compound has no charged side chain, yet stillpossesses appreciable antagonist potency compared to arginin at thisposition.

Position “X” may be —(CH₂)_(n)NH— or —(CH₂)_(n)—S—, where n is aninteger from 1 to 4, preferably 2 or 3, —(CH₂)₂O—, —(CH₂)₃O—, —(CH₂)₃—,—(CH₂)₄—, —CH₂COCHRNH—, or —CH₂—NHCOCHRNH— where R is the side chain ofany common or uncommon L- or D-amino acid.

This group provides the cyclization link, for example between the Argand Phe residues of compound 1, and thus influences the structure of thecyclic backbone. In addition, substituents such as R on this linkercould potentially interact with receptor residues to increase affinityof the antagonists.

N-methylation of the amino acid components of the cycles tends to reducethe receptor binding affinity and antagonist potency of the compounds(e.g., 64, 65), although N-methylation of the delta nitrogen ofornithine has virtually no effect on antagonist potency.

Multiple changes on the scaffold can be detrimental to obtainingincreased antagonist potency relative to 1. Thus although L-Phe was asuitable replacement (e.g., 60) for L-Trp in 1, with little change inantagonist potency, a combination of changes to 1, such as L-Phe for Trpand either L-HomoPhe for Arg (e.g., 67) or p-chloro-phenylalanine forArg (e.g., 68), led to reduced affinity for the receptor and reducedantagonist potency. Similarly when L-Trp in 1 was replaced by L-Phe andD-Cha was also replaced by D-Phe, the compound lost substantial potency(e.g., 62). While a change from D-Cha in 1 to D-Phe led to retention ofthe antagonist potency (e.g., 33), this change is detrimental whencoupled with replacement of L-Trp by L-Phe (62).

Clearly there is cooperativity in the binding of these residues to thereceptor, since either of the single changes (e.g., 33, 60) results insubstantially higher potency than when the changes are made together(e.g., 62). When the Arg was replaced by an aromatic group stillcontaining a charged amine (69), there was a significant loss inactivity, as was observed when Phe of 60 was replaced by a substitutedtyrosine (70).

All these changes are indicative of what can and cannot be tolerated onthe cyclic scaffold used to engender affinity for the human PMN C5areceptor and antagonist potency. It is recognised that C5a receptors onother types of cells may have different tolerances for side chains, butthe cyclic scaffold will still form the basis of active compounds.

Example 5 Reverse Passive Arthus Reaction in the Rat

A reverse passive peritoneal Arthus reaction was induced as previouslydescribed (Strachan et al., 2000), and a group of rats were pretreatedprior to peritoneal deposition of antibody with AcF-[OPdChaWR] (1) byoral gavage (10 mg kg⁻¹ dissolved in 10% ethanol/90% saline solution toa final volume of 200 μL) or an appropriate oral vehicle control 30 minprior to deposition of antibody. Female Wistar rats (150-250 g) wereanaesthetised with ketamine (80 mg kg⁻¹ i.p.) and xylazine (12 mg kg⁻¹i.p.).

The lateral surfaces of the rat were carefully shaved and 5 distinctsites on each lateral surface clearly delineated. A reverse passiveArthus reaction was induced in each dermal site by injecting Evans blue(15 mg kg⁻¹ i.v.), chicken ovalbumin (20 mg kg⁻¹ i.v.) into the femoralvein 10 min prior to the injection of antibody. Rabbit anti-chickenovalbumin (saline only, 100, 200, 300 or 400 μg antibody in a finalinjection volume of 30 μL) was injected in duplicate at two separatedermal sites on each lateral surface of the rat, giving a total of 10injection sites per rat. Rats were placed on a heating pad, andanaesthetic was maintained over a 4 h-treatment period with periodiccollection of blood samples. Blood was allowed to spontaneously clot onice, and serum samples were collected and stored at −20° C. Four hoursafter induction of the dermal Arthus reaction, the anaesthetised rat waseuthanased and a 10 mm² area of skin was collected from the site of eachArthus reaction. Skin samples were stored in 10% buffered formalin forat least 10 days before histological analysis using haematoxylin andeosin stain. Additionally, a second set of skin samples were placed in 1mL of formamide overnight, and the absorbance of Evans blue extractionmeasured at 650 nm, as an indicator of serum leakage into the dermis.FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the optical density ofdermal punch extracts following intradermal injection of rabbitanti-chicken ovalbumin at 0-400 μg site⁻¹ following pretreatment withAcF-[OPdChaWR] intravenously, orally or topically. Data are shown asabsorbance at 650 nm as a percentage of the plasma absorbance, as meanvalues±SEM (n=3-6). *indicates a P value ≦0.05 when compared to Arthuscontrol values.

Rats were pretreated with the C5aR antagonist, AcF-[OPdChaWR] (1) as theTFA salt, either intravenously (0.3-1 mg kg⁻¹ in 20 μL saline containing10% ethanol, 10 min prior to initiation of dermal Arthus), orally(0.3-10 mg kg⁻¹ in 200 μL saline containing 10% ethanol by oral gavage,30 min prior to initiation of dermal Arthus in rats denied food accessfor the preceding 18 hours) or topically (200-400 μg site⁻¹ 10 min priorto initiation of dermal Arthus reaction), or with the appropriatevehicle control. Topical application of the antagonist involvedapplication of 20 μL of a 10-20 mg mL⁻¹ solution in 10% dimethylsulphoxide (DMSO), which was then smeared directly onto the skin at eachsite, 10 min prior to induction of the Arthus reaction.

The saline-only injection site from rats treated with Evans blue onlyserved as antigen controls, the saline-only injection site from ratstreated with Evans blue plus topical DMSO only served as a vehiclecontrol, the saline-only injection site from rats treated with Evansblue plus either intravenous, oral or topical antagonist only served asantagonist controls, and Evans blue plus dermal rabbit anti-chickenovalbumin served as antibody controls. Topical application of thepeptide AcF-[OPGWR] which has similar chemical composition andsolubility to that of AcF-[OPdChaWR] (1), but with an IC₅₀ bindingaffinity of >1 mM in isolated human PMNs, served as an inactive peptidecontrol. AcF-[OPGWR] was also dissolved in 10% DMSO and appliedtopically at 400 μg site⁻¹ 10 min prior to initiation of the Arthusreaction.

TNF-α Measurement

Serum TNF-α concentrations were measured using an enzyme-linkedimmunosorbent assay (ELISA) kit. Antibody pairs used were a rabbitanti-rat TNF-α antibody coupled with a biotinylated murine anti-ratTNF-α antibody. FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the serumTNF-α concentrations at regular intervals after initiation of a dermalArthus reaction, with group of rats pretreated with AcF-[OPdChaWR]intravenously, orally or topically. Data are shown as mean values±SEM(n=3-6). *indicates a P value of ≦0.05 when compared to Arthus controlvalues.

Interleukin-6 Measurement

An ELISA method as described previously was used to measure serum andperitoneal lavage fluid interleukin-6 (IL-6) concentrations (Strachan etal., 2000).

Pathology Assessment

Rat skin samples were fixed in 10% buffered formalin for at least 10days, and stained with haematoxylin and eosin using standardhistological techniques. Dermal samples were analysed in a blind fashionfor evidence of pathology, and the degree of rat PMN infiltration wasscored on a scale of 0-4. Initiation of a dermal Arthus reactionresulted in an increase in interstitial neutrophils, which as quantifiedin the following manner. Sections were given a score of 0 if noabnormalities were detected. A score of 1 indicated the appearance ofincreased PMNs in blood vessels, but no migration of inflammatory cellsout of the lumen. A score of 2 and 3 indicated the appearance ofincreasing numbers of PMNs in the interstitial tissue and more prominentaccumulations of inflammatory cells around blood, vessels. A maximalscore of 4 indicated severe pathological abnormalities were present indermal sections, with excessive infiltration of PMNs into the tissuesand migration of these cells away from blood vessels. FIG. 3A, FIG. 3B,FIG. 3C, and FIG. 3D show the intradermal injection of increasingamounts of antibody leads to a dose-responsive increase in the pathologyindex scored by dermal samples (FIG. 3A). Data are shown for dermalsamples intradermally injected with saline or 400 μg site⁻¹ antibody(n=5) in rats pretreated with AcF-[OPdChaWR] intravenously (FIG. 3B)(n=3), orally (FIG. 3C) (n=3) and topically (FIG. 3D) (n=3). Data areshown as mean values±SEM. * P≦0.05 when compared to Arthus values usinga non-parametric t-test.

Example 6 Effect of C5a Antagonist on Model of IntestinalIschemia-Reperfusion Injury in Rats

Female Wistar rats (250-300 g, n=132) were fasted and given only waterfor 12 h prior to all experiments. Animals were anaesthetised by theintraperitoneal injection of 80 mg kg⁻¹ ketamine and 10 mg kg⁻¹ xylazineand throughout the procedure rats were placed on a heating pad tomaintain normal body temperature. The abdomen was opened through amidline incision to expose the superior mesenteric artery (SMA). Anon-traumatic occlusive device for the artery was fashioned from a silksuture looped though a length of polyethylene tubing. The SMA wasoccluded by applying traction to the ends of the loop. A polyethylenecatheter was inserted in the femoral vein to allow the infusion ofeither 1 mg kg⁻¹ AcF-[OPdChaWR] (1) in 5% ethanol or sterile,pyrogen-free saline in 0.2 mL volume. Infusions were made over a 2-minperiod. Oral dosing of AcF-[OPdChaWR] (1) (0.3, 1, 10 mg kg⁻¹) wasachieved by gavage (0.2 mL saline in 25% ethanol) 60 min prior to SMAocclusion. Intestinal ischemia was induced by clamping the SMA for 30min, after which the occlusive suture was removed, then reperfusion wasmonitored for another 120 min. Sham-operated rats were treated in anidentical fashion, with the omission of vascular occlusion, and wereinfused with 0.2 mL of sterile, pyrogen-free saline or gavaged with 0.2mL of saline in 25% ethanol. Blood samples (50 μL) were collected intoheparinised Eppendorf tubes at regular intervals over the 180-minduration of the experiments for the estimation of PMN numbers. In adifferent series of identical experiments, whole blood was collected atregular intervals over the 180 min and allowed to clot on ice, and serumor plasma samples collected and stored at −20° C. for later measurementof tumour necrosis factor-α (TNF-α), haptoglobin (Hp) and aspartateaminotransferase (AST) concentrations. At the end of the 120 minreperfusion period, the animals were euthanased with an overdose ofpentobarbital. A section of the occluded ileum was removed, the lumenrinsed with saline, and the intestine was blotted dry, then weighed.Specimens were dried in an oven for 24 hours at 80° C. to obtain thetissue dry weight. Intestinal oedema was determined by assessing the wetand dry tissue weight ratio. Additionally, segments of both ischaemicand normal intestine were harvested and rinsed with saline andimmediately fixed in 10% buffered formaldehyde-saline for histologicalstudies.

FIG. 4 shows that the wet-to-dry weight ratio of the small intestine issignificantly elevated after ischemia-reperfusion compared tosham-operated animals. Treatment with the C5a receptor antagonistAcF-[OPdChaWR] 1 mg/kg i.v. and 10 mg/kg p.o. significantly reducedtissue edema compared to untreated ischemia-reperfusion (I/R) animals.Data are shown as means±SEM (n=4-6 in each group). *, P<0.05 vs.sham-operated animals. +, P<0.05 vs. I/R animals.

Neutropenia Assay

Blood (50 μL) for PMW was placed into heparinised tubes and then layeredover an equal volume of Histopaque 1083 (Sigma, U.S.A.), PMNs wereisolated and cell number counted on a haemocytometer. Concentrations ofPMNs values are presented as mean percentage±SEM of the values obtainedimmediately prior to SMA occlusion.

FIG. 5A and FIG. 5B show that the gut ischemia-reperfusion causedsignificant reduction in circulating PMN concentrations compared withsham-operated animals (FIG. 5A and FIG. 5B). Pretreatment of rats withC5a receptor antagonist AcF-[OPdChaWR] 1 mg/kg i.v. (FIG. 5A) and 10mg/kg p.o. (FIG. 5B) significantly inhibited ischemia-reperfusioninduced neutropenia. 1 mg/kg p.o, (FIG. 5B) treated animals showed noinhibition of neutropenia. Data are shown as means±SEM (n=6 in eachgroup). *equals P<0.05 vs. Control animals. Bar shows 30-min period ofischemia.

Tumor Necrosis Factor-α Measurement

Serum TNF-α concentrations were measured using an enzyme-linkedimmunosorbent assay, (Pharmingen, USA), according to the manufacturersinstructions. Concentrations of TNF-α in serum samples were determinedby linear regression analysis from the standard curve.

FIG. 6 shows that the gut ischemia-reperfusion resulted in significantelevation in serum TNF-α compared with sham-operated animals.Pre-treatment of rats with the C5a receptor antagonist AcF-[OPdChaWR] 1mg/kg i.v. and 1-10 mg/kg p.o. completely inhibited the change in serumTNF-α levels. Data are shown as means±SEM (n=6 in each group). *=P<0.05vs. sham-operated animals. The bar shows 30-min period of ischemia.

Haptoglobin Assay

Serum Hp was measured by using an Hp assay kit (Tridelta. Development,Ltd., U.K.), according to the manufacturers' instructions.Concentrations of Hp in the serum samples were determined by linearregression analysis from the standard curve.

FIG. 7 shows that gut ischemia-reperfusion resulted in a significantincrease in serum haptoglobin compared to sham-operated animals.Pretreatment of rats with C5a receptor antagonist AcF-[OpdChaWR] 1 mg/kgi.v. and 1, 10 mg/kg p.o. significantly inhibited the increase in serumhaptoglobin levels. Data are shown as means±SEM (n=4-6 in each group).*, P<0.05 vs. sham-operated animals. +, P<0.05 vs. I/R animals.

Aspartate Aminotransferase Assay

Plasma AST (AST/GOT; Sigma, USA) concentrations were measured accordingto manufacturer's instructions within 48 h of collecting plasma. PlasmaAST concentrations were derived from calibration curve. Results areexpressed in Sigma-Franke (SF) units/ml.

FIG. 8 shows that the gut ischemia-reperfusion resulted in a significantincrease in plasma aspartate aminotransferase compared to sham-operatedanimals. Treatment with the C5a receptor antagonist AcF-[OPdChaWR] 1mg/kg i.v. and 1, 10 mg/kg significantly reduced gutischemia-reperfusion induced aspartate aminotransferase compared tountreated I/R animals. Data are shown as means±(n=6 in each group). *,P<0.05 vs. sham-operated animals. +, P<0.05 vs. I/R animals.

Histopathology

Specimens were fixed in 10% formaldehyde-saline, embedded in paraffinwax, sectioned serially, and stained with haematoxylin and eosin.Tissues were read and scored by a trained observer in a blinded fashion.The degree of intestinal tissue injury was determined using a previouslydescribed graded scale ranging from 0-8 (Chiu et al., 1970).

FIG. 9 shows that gut ischemia-reperfusion resulted in significantdamage to the intestine compared to sham-operated animals. Treatmentwith C5a receptor antagonist AcF-[OPdChaWR] 1 mg/kg i.v. and 1, 10 mg/kgsignificantly reduced gut ischemia-reperfusion induced tissue damagecompared to untreated I/R animals. Data are shown as mean±SEM (n=6 ineach group). *, P<0.05 vs. I/R animals.

Example 7 Rat Monoarticular Antigen-Induced Arthritis

Female Wistar rats (150-250 g) were obtained from the Central AnimalBreeding House, University of Queensland. Methylated bovine serumalbumin (mBSA) (0.5 mg) was dissolved in Freund's complete adjuvant (0.5mg) and sonicated to produce a homogenous suspension. Each rat receiveda subcutaneous injection of this suspension (0.5 mL) on days 1 and 7. Onday 12-28, rats were separated into separate cages, and body weight andfood and water intake monitored daily. Rats received either ordinary tapwater or drinking water containing AcF-[OPdChaWR] (1). Body weight andwater intake were monitored daily, and rats received a daily dose of 1mg/kg/day of the C5aR antagonist AcF-[OPdChaWR] (1) for days 12-28 ofthe trial. On day 14, rats were anaesthetised and their hind limbsshaved. Each rat received an intra-articular (100 μL) injection of mBSA(0.5 mg) in the left knee, and saline in the right knee. The saline-onlyknee from rats receiving normal drinking water served as a salinecontrol, the saline only knee from rats receiving AcF-[OPdChaWR] (1) inthe drinking water served as an antagonist control.

Rats were euthanased on day 28, and whole blood collected into anEppendorf tube and allowed to clot on ice. Blood samples werecentrifuges (11,000 rpm×3 min) and serum collected and stored at −20° C.until analysis of serum cytokines using an ELISA. Each knee capsule waslavaged with 100 μL saline, and the total cell count determined using ahaemocytometer. In addition, an aliquot of the knee joint lavage fluidwas dropped onto a glass slide, and allowed to air dry. Once dry, cellswere stained with a differential stain (Diff Quick) and a differentialcell count was performed using a 40× dry lens microscope. The remaininglavage fluids from each joint were stored at −20° C. until lateranalysis of intra-articular cytokine levels using an ELISA. Each kneejoint was removed and the skin was split with a scalpel blade to allowfixation. Knee samples were stored in 10% buffered formalin for ≧10 d.Knees were then rinsed with distilled water and placed in a saturatedsolution of EDTA solution for 21 d for decalcification before beingembedded in paraffin wax.

Knee tissue samples were prepared using standard histological techniquesas described above in Example 6 and stained using an heamotoxylin andeosin stain. Histological slides were analysed in a blinded fashion.Tissue sections were scored from 0-4, with a score of 0 indicating noabnormalities, and increasing scores with the appearance of synovialcell proliferation, inflammatory cell infiltration, cartilagedestruction and haemorrhage. In no samples was there evidence ofsignificant bone erosion. Samples of serum and synovial fluid werethawed on the day of ELISA analysis for TNF-α and IL-6 levels.Concentrations were determined from a standard curve, using an ELISA asdescribed previously in Example 6.

FIG. 10 shows the inhibition of arthritic right knee joint swelling byAcF-[OPdChaWR] given orally on Days-2 to +14, while FIG. 11 shows theinhibition of right knee joint TNF-α; and IL-6 levels in joint lavage.Untreated refers to animal not treated with AcF-[OPdChaWR] but withright knee challenged with antigen following sensitisation.

Example 8 Topical Dermal Administration of C5a Antagonists

The invention teaches that topical administration of C5a antagonists maybe used for the treatment of topical inflammatory involving activationof the complement system. In this example we demonstrate that topicalapplication of C5a antagonists can also result in systemicpharmacological actions and the appearance of pharmacologically relevantconcentrations of C5a antagonists in the circulation.

The in vivo pharmacological properties of cyclic antagonists wereexamined following topical dermal administration. A model ofendotoxaemia was used, in which 1 mg/kg Escherichia coli liposaccharide(LPS; serotype 55: B5, Sigma, USA, stored at 100 mg mL⁻¹ in dH₂O, 4°C.), was injected i.v. into a rat, resulting in acute changes incirculating PMN levels and blood pressure. These parameters weremeasured in the presence and absence of C5aR antagonists (1 mg/kg i.v.or 50 mg/kg/rat topically). This study shows that topical administrationof C5a receptor antagonists is an effective method of delivery of thesecompounds, with systemic pharmacological activity being observed.

Female Wistar rats weighing between 200-250 g were used for in vivotesting of all C5a receptor antagonists. Rats were anaesthetized usingthe procedure described above, and transferred on to a heating pad toensure that body temperature was maintained throughout the experiment. Acatheter was inserted in the femoral vein, secured with a suture andflushed with 100 μL of heparinised saline. Rats were dosed with eitherthe antagonist or vehicle, intravenously (1 mg kg⁻¹ in 200 μL salinecontaining 10% ethanol, 10 min prior to LPS challenge) or topically (10mg site-1 in 50% dimethyl sulphoxide/H₂O 60 min prior to complementchallenge). Rats were infused i.v. via the femoral catheter with eitherLPS (1 mg kg⁻¹ i.v. in 100 μL saline), recombinant human C5a (2 μg kg⁻¹in 100 μL), or vehicle control 10 minutes after the iv administration or60 min after topical administration, of antagonist or vehicle control.All agents infused i.v. were injected over a 2 min period, and werefollowed with a subsequent injection of 100 μL of saline to ensurecomplete delivery of the drug.

Whole blood samples (0.1 mL) were collected from the tail vein at thetime of drug and LPS or C5a administration. Samples were collected at−15, 0, 5, 10, 15, 30, 60, 90, 120 and 150 min relative to the injectionof LPS (zero time), and placed in tubes containing heparin (500Units/mL). To isolate PMNs, 100 μL of blood was layered on to 200 μL ofHistopaque 1077 (Sigma U.S.A.) solution and centrifuged at 400×g for 30min min at room temperature (25° C.). The supernatant layer ofplatelet-rich plasma, the monocyte and lymphocyte interface and theseparation layer of Histopaque were removed and discarded, leaving thePMN and red blood-cell rich layer. 9 mL of cold distilled water (4° C.)was added to the remaining pellet and shaken for 40 sec to lyse the redblood cells. Dulbecco's Phosphate-buffered saline (10× concentration),was added to restore isotonicity. The cells were then centrifuged at400×g for 15 min at 10° C. The resulting supernatant was discarded,leaving a pellet of PMNs. The PMNs were further washed in 9 mL ofsaline, and centrifuged again at 400×g for 10 min at 10° C. Again theresulting supernatant was discarded, and the remaining pellet wasresuspended in 100 μL or saline and mixed well. The number of PMNS wascounted on a haemocytometer. The number of cells at each time point wascalculated as a percentage of the total number of cells at time zero,prior to complement challenge or LPS.

The female Wistar rats chosen for this study were divided into thefollowing treatment groups:

(a) LPS alone, in which 100 μL 5% ethanol (−15 min)+1 mg/kg LPS (0 min)were infused;

(b) ethanol control, in which 100 μL 5% ethanol was infused at −15 minto examine the influence of ethanol on the parameters measured in therat;

(c) antagonist control, in which 1 mg/kg of cyclic antagonist wasinfused i.v. at −15 min;

(d) i.v. antagonist+LPS, in which 1 mg/kg cyclic antagonists (−15 min)and 1 mg/kg LPS (0 min) were infused; and

(e) topically-applied antagonist+LPS, in which 10 mg/rat of antagonistwas smeared on the abdominal area of the rat, followed by i.v. infusionof LPS at 0 min.

Administration of C5a antagonists such as 3D53 (PMX53) to the dermis ofrats in an applied dose of 50 mg/kg results in the detection ofpharmacologically significant levels of the drug in the circulatingplasma. The application of the drug in a variety of solvents, such asdimethyl sulphoxide (DMSO), propylene glycol (PG) and water, in variouscombinations leads to the appearance of pharmacologically-relevantconcentrations of the drug in the circulation, as illustrated in FIG.12. This shows that dermal application of 3D53 in DMSO/distilled H₂O orpropylene glycol (PG)/H₂O results in the appearance of the C5aantagonist in the circulating plasma within 15 min, and that significantlevels persist for at least 4 hr.

Example 9 Systemic Effects of C5a Antagonists Following DermalApplication

As described in Example 8, topical application of C5a antagonist to thedermis of an animal results in pharmacologically-relevant levels of thedrug in the circulation. To show that these levels have systemicactivity, the ability of these circulating levels of C5a antagonist toinhibit the neutropenic effects of C5a administered i.v. was determined.

The C5a receptor antagonist 3D53 was administered (10 mg/per rat) in 50%propylene glycol and 50% distilled H₂O. The composition was smearedevenly over the abdominal skin (4×8 cm²) for 30 minutes, then C5a wasadministered i.v. in a dose of 2 μg/kg in 200 μL saline solution. Bloodsamples were taken at time points: −30 (before drug administration), 0(before injection of C5a) and 5, 15, 30, 60, and 120 minutes fordetermination of circulating PMNs. As shown in FIG. 13, topicaladministration of compound 3D53 did indeed prevent the neutropaeniceffects of C5a.

Example 10 Topical Administration of C5a Antagonists Inhibits theSystemic Effects of LPS

The C5a antagonists, 3D53 (compound 1), as well as compound 17 andcompound 45 were applied topically at a dose of 10 mg/rat in 50%DMSO/50% H₂O, as described above. LPS was injected 1 mg/kg i.v. 60 minafter dermal application of the antagonists. Circulating PMN levels weremonitored for 150 min following LPS injection, and the percentage changein PMN levels from zero time, when LPS was injected, was calculated. Theresults, illustrated in FIG. 14A, FIG. 14B, and FIG. 14C, show that eachC5a antagonist applied topically inhibited the neutropenia response toi.v. LPS, and that the inhibition was comparable to that observedfollowing i.v. administration of the drugs.

Example 11 Effect of C5a Antagonist on Ischemia-Reperfusion Injury

Lower limb ischemia-reperfusion (I/R) injury is a serious problemfollowing the surgical repair of abdominal aortic aneurysm, as well asfollowing traumatic crush injuries (Kerrigan and Stotland, 1993).Ischemia and the subsequent reperfusion of the skeletal muscle tissuestimulates an inflammatory response in the affected muscle, as well asinducing injury in other tissues (Gute et al, 1998). In severe cases oflimb ischemia, the resulting reperfusion is associated with highmortality, resulting from multiple system organ failure (Defraigne andPincemail, 1997). In order to investigate the capacity of a potent C5areceptor antagonist to inhibit various parameters of local and remoteorgan injury following lower limb ischemia-reperfusion (I/R) in rats,rat hindlimbs were subjected to 2 hours ischemia and 4 hoursreperfusion. This tourniquet shock model has been widely used as a modelof lower limb I/R injury.

Rats were subjected to 2 hours bilateral hindlimb ischemia and 4 hoursreperfusion. Drug-treated rats received AcF-[OPdChaWR] (1 mg/kg) i.v.either 10 min before ischemia or 10 min prior to reperfusion, or orally(10 mg/kg) 30 min prior to ischemia. Levels of circulating creatinekinase (CK), lactate dehydrogenase (LDH), alanine and aspartateaminotransferase (ALT/AST), creatinine, blood urea nitrogen (BUN),polymorphonuclear leukocytes (PMNs) and calcium (Ca⁺⁺) and potassium(K⁺) ions were determined. These biochemical indices are known toreflect tissue or organ injury following I/R events. Other parametersmeasured included urinary protein levels, muscle edema andmyeloperoxidase (MPO) concentrations in the lung, liver and muscle alongwith liver homogenate TNF-α concentrations. No significant changes wereobserved in any of these markers compared to sham-operated animals,indicating that the drug alone had no adverse effects as defined bychanges in these markers. Limb I/R injury was characterized bysignificant elevations of CK, LDH, ALT, AST, creatinine, BUN,proteinuria, PMNs, serum K⁺, muscle edema, organ MPO and liverhomogenate TNF-α concentrations, but a significant reduction in serumCa⁺⁺ concentrations. When rats were treated with AcF-[OpdChaWR], therewere significant improvements in all these parameters.

The study was performed in accordance with guidelines from the NationalHealth & Medical Research Council of Australia, and the experimentalprotocol approved by the University of Queensland Animal EthicsCommittee. Female Wistar rats weighing 250-300 g were fasted overnightbefore being anaesthetized with the i.p. injection of 6 mg/kg xylazineand 120 mg/kg ketamine. Anesthesia was maintained throughout the studyby additional injections of ketamine. Rats were placed on a heating padto maintain normal body temperature, and a polyethylene catheter wasinserted into the right jugular vein for the infusion of the C5aantagonist or 7% ethanol/saline. Bilateral hindlimb ischemia was theninduced through the application of latex o-rings (marking rings; HayesVeterinary Supplies, Brisbane, Australia) above the greater trochanterof each hind limb. Following 2 hours of ischemia, the latex rings werecut and removed and limbs allowed to reperfuse for 4 hours. Sixexperimental groups were used:

(a) sham-operated,

(b) ischemia-only,

(c) I/R-only,

(d) I/R+C5a antagonist (1 mg/kg, i.v.) administered 10 min prior toischemia,

(e) I/R+C5a antagonist (10 mg/kg, p.o.) administered 30 min prior toischemia, and

(I/R+C5a antagonist (1 mg/kg, i.v.) administered 10 min prior toreperfusion.

Sham-operated animals did not undergo any ischemia or reperfusion, andischemia-only animals had tourniquets applied for 2 hours withoutsubsequent reperfusion. All other groups underwent 2 hours of ischemiaand 4 hours of reperfusion. Sham-operated, ischemia-only and I/R groupswere infused with 7% ethanol/saline 10 min prior to ischemia, instead ofdrug. Blood was collected throughout the study from the tail vein, andserum or plasma was stored at either 4° C. or −20° C. for laterbiochemical assays. Urine was collected over the last hour of the studyfor the determination of urinary protein levels. At the completion ofthe experiment, rats were euthanased, and sections of the lungs, liverand gastrocnemius muscle removed and weighed for edema, neutrophilaccumulation and liver TNF-α studies.

All experimental results are expressed as means standard error of themean (SEM). Data analysis was performed using GraphPad Prism 3.0software (GraphPad Software, Inc. USA). Statistical comparisons weremade to sham-operated and I/R-only groups, using a one-way analysis ofvariance followed by a Dunnett comparison post-test analysis.Statistical significance was assessed at P<0.05.

(a) Inhibition of Creatine Kinase and Lactate Dehydrogenase

Circulating levels of creatine kinase (CK) were measured in serumsamples taken immediately after ischemia, following 1, 2 and 3 hoursreperfusion, and at the completion of the study using a CK kit (Sigma,St. Louis, USA) according to the manufacturer's instructions. Serum wasalso taken 10 min after tourniquet release for CK measurement inI/R-only animals. A 1:5 dilution was used for samples taken after 2hours reperfusion.

Circulating levels of lactacte dehydrogenase (LDH) were measured inserum samples taken immediately after ischemia, following 1, 2 and 3hours reperfusion, and at the completion of the study. Serum was alsotaken 10 min after tourniquet release for LDH measurement in I/R-onlyanimals. Concentrations of LDH were determined with a LDH kit (Sigma),according to the manufacturer's instructions, with a 1:4 dilution ofsamples taken after 2 hours reperfusion. For both enzymes, all sampleswere stored at 4° C. and analyzed within 24 hours of collection. Resultswere expressed as Sigma-Franke (SF) units/mL.

As illustrated in FIG. 15A and FIG. 15B, bilateral hindlimb I/R resultedin elevation of circulating levels of both CK and LDH after 1, 2, 3 and4 hours of reperfusion, with peaks of both enzymes reached after 4hours. Ischemia-only rats showed no significant elevation of either CKor LDH levels (CK, 58.3±23.5 units/mL; LDH, 269.5±72.8 units/mL; P>0.05;n=4) compared to sham-operated rats. In I/R-only rats there was nosignificant increase in the levels of these enzymes 10 min aftertourniquet release (CK, 73.9±28.1 units/mL; LDH, 395.3±123.2 units/mL;P>0.05; n=4), compared to sham-operated rats, indicating areperfusion-dependent elevation over the 4 hour time period. Reperfusionsignificantly elevated the plasma levels of both CK and LDH (P<0.05).Rats treated prior to ischemia with the C5a antagonist, either i.v. (1mg/kg) or orally (10 mg/kg), had similar significantly decreased levelsof both CK and LDH compared to I/R-only rats (P<0.05). In addition, ratstreated i.v. with the C5a antagonist (1 mg/kg) just prior to reperfusionalso displayed significant inhibition of CK and LDH levels, of similarmagnitude to pre-ischemia treated rats (P<0.05). Levels of these enzymesduring reperfusion in all the drug-treated rats were significantlyhigher than in sham-operated rats, indicating partial inhibition by theC5a antagonist (P<0.05).

(b) Inhibition of Alanine Transaminase and Aspartate Aminotransferase

Circulating levels of alanine aminotransferase (ALT) and aspartateaminotransferase (AST) were measured in plasma samples taken at thecompletion of the study and following 2 and 3 hours reperfusion. Serumwas also taken 10 min after tourniquet release for measurement of andAST in I/R-only animals. Concentrations of ALT and AST were determinedwith an ALT/AST kit (GPT/GOT; Sigma), according to the manufacturer'sinstructions, within 24 hours of collecting plasma, which was stored at4° C. Results were expressed as SF units/mL.

As shown in FIG. 16A and FIG. 16B, limb I/R resulted elevation of ALTand AST in the plasma after 2, 3 or 4 hours of reperfusion, with peaksof both enzymes reached after 4 hours. Rats receiving 2 hours ofischemia alone showed no significant elevation of either ALT or ASTlevels (ALT, 12.9±4.6 unit/mL; AST, 91.4±10.4 units/mL; P>0.05; n=4)compared to sham-operated rats. In I/R-only rats, 10 min aftertourniquet release, there was no significant increase in levels of theseenzymes (ALT, 18.1±4.4 units/mL; AST, 105.9±21.3 units/mL; P>0.05; n=4),compared to sham-operated rats, again indicating a reperfusion-dependentelevation. C5a antagonist-treated rats in all 3 groups were found tohave similar significant decreases in ALT and AST levels compared toI/R-only rats (P<0.05). After 4 hours of reperfusion, but not at 2 or 3hours, drug-treated rats had significantly increased levels of ALTcompared to sham-operated rats (P<0.05; FIG. 16A). In contrast, thesedrug-treated rats had increased AST levels compared to sham-operatedrats at all time points measured, indicating the differential inhibitionof the C5a antagonist for ALT and AST (P<0.05; FIG. 16B).

(c) Inhibition of Changes in Serum Levels of Potassium and Calcium Ions

Serum levels of potassium ion (K⁺) were measured with a flame photometer(Corning 435; Corning, U.S.A.) after the completion of each experiment,and 10 min after tourniquet release for I/R-only animals. Serum calciumion (Ca⁺⁺) concentrations were also measured at the completion of thestudy and 10 min after tourniquet release for I/R-only animals, using acalcium kit (Sigma). Samples were stored at −20° C., and analyzed for K⁺and Ca⁺⁺ levels within 2 weeks of collection. Results were expressed asmmol/L, and are summarized in Table 4.

TABLE 4 Alterations in serum cation levels following ischemia and 4hours reperfusion in rats Serum K⁺ Serum Ca⁺⁺ Experimental Group n^(a)(mmol/L) (mmol/L) Sham-operated 8 4.84 ± 0.29* 2.66 ± 0.06*Ischemia-only 4 4.70 ± 0.17* 2.52 ± 0.10* I/R^(b)-only 10 7.53 ±0.34^(†) 2.23 ± 0.09 I/R + 1 mg/kg 8 6.13 ± 0.18* 2.46 ± 0.04* i.v.pre-ischemia I/R + 10 mg/kg 6 5.57 ± 0.58* 2.49 ± 0.04* p.o.pre-ischemia I/R + 1 mg/kg 6 5.02 ± 0.32* 2.45 ± 0.10* i.v.pre-reperfusion Data represent the mean ± SEM. ^(a)Number of rats^(b)Ischemia/reperfusion *P < 0.05 vs. I/R-only ^(†)P < 0.05 vs.sham-operated

Inhibition of Blood Urea Nitrogen, Creatinine and Urinary Protein

Circulating levels of blood urea nitrogen (BUN) were measured in serumsamples taken at the completion of the study using a urea nitrogen kit(Sigma) according to the manufacturer's instructions. Samples werestored at −20° C. and were analysed within 2 weeks of collection.Circulating levels of creatinine were measured in serum samples taken atthe completion of the study using a creatinine kit (Sigma) according tothe manufacturer's instructions. Protein concentrations in urine samplescollected over a 1 hour period prior to the completion of the study weredetermined with a protein kit (Sigma) according to the manufacturer'sinstructions. Samples were stored at 4° C. and analyzed within 24 hoursof collection. Results for all three parameters, expressed as mg/dL, areshown in Table 5.

TABLE 5 Alterations in kidney injury markers following ischemia and 4hours reperfusion in rats Plasma Serum BUN^(b) Creatinine ProteinuriaExperimental Group n^(a) (mg/dL) (mg/dL) (mg/dL) Sham-operated 8 21.9 ±1.2* 0.79 ± 0.12*  10.7 ± 2.2* Ischemia-only 4 22.8 ± 2.5* 0.62 ± 0.26* 13.3 ± 7.0* I/R^(c)-only 10 41.9 ± 3.1^(†) 1.66 ± 0.09^(†) 120.3 ±18.9^(†) I/R + 1 mg/kg i.v. 8 23.6 ± 1.6* 1.07 ± 0.07*  36.4 ± 11.4*pre-ischemia I/R + 10 mg/kg p.o. 6 21.8 ± 2.1* 1.14 ± 0.14*  45.8 ± 9.8*pre-ischemia I/R + 1 mg/kg i.v. 6 22.0 ± 2.2* 1.22 ± 0.05*  41.2 ± 11.4*pre-reperfusion Data represent the mean ± SEM. ^(a)Number of rats^(b)Blood urea nitrogen ^(c)Ischemia/reperfusion ^(d)Not detectable *P <0.05 vs. I/R-only ^(†)P < 0.05 vs. sham-operated

Hyperkalaemia was observed in I/R-only rats compared to sham-operatedrats after 4 hours of reperfusion (P<0.05). Rats in all 3 drug-treatmentgroups had significantly lower K⁺ levels than I/R-only rats, with ratstreated just prior to reperfusion showing near-normal levels (P<0.05).Following ischemia and 4 hours of reperfusion, I/R-only rats hadsignificantly decreased serum concentrations of Ca⁺⁺ compared tosham-operated animals (P<0.05). Rats in all 3 drug-treatment groupsshowed a similar inhibition of the FR-induced decrease in Ca⁺⁺ levels(P<0.05). Levels of both K⁺ and Ca⁺⁺ in drug-treated I/R rats, as wellas ischemia-only rats, were not significantly different from those insham-operated rats (P>0.05). Levels of these ions 10 min after therelease of the tourniquet in I/R-only rats (K⁺, 5.27±0.49 mmol/L; Ca⁺⁺,2.50±0.17 mmol/L; P<0.05; n=4) were also not significantly differentfrom those in sham-operated rats.

Following ischemia and 4 hours reperfusion, I/R-only rats hadsignificantly elevated serum BUN and creatinine levels, as well asincreased urinary protein concentrations, compared to sham-operated rats(P<0.05). Two hours of ischemia alone caused no increase in any of theseparameters compared to sham-operated rats (P>0.05). Rats treated withthe C5a antagonist in all 3 groups had significantly lower levels ofthese parameters compared to I/R-only rats (P<0.05), and these levelswere not significantly different from those in sham-operated rats(P<0.05).

Inhibition of Polymorphonuclear Leukocyte Numbers and NeutrophilAccumulation

The numbers of circulating PMNs were measured in heparinised bloodsamples taken just prior to ischemia and at the completion of the study,as described by Strachan et al., (2000). Numbers of PMNs in the finalsamples were expressed as a mean percentage SEM of pre-ischemia numbers.

As shown in FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D, the number ofcirculating PMNs was found to be significantly elevated in I/R-only ratsfollowing 4 hours of reperfusion, compared to sham-operated rats(P<0.05). Ischemia-only rats had no significant elevation of PMNscompared to sham-operated rats (P>0.05). PMN numbers in rats from all 3drug-treated groups were not significantly different from those insham-operated rats (P>0.05), and were significantly decreased comparedto those in I/R-only rats (P<0.05), with rats treated i.v. (1 mg/kg)pre-ischemia displaying the greatest inhibitory effect.

The infiltration of neutrophils into the liver, lung and muscles of ratswas determined by measuring the level of myeloperoxidase (MPO) activity.Sections of lung (˜0-5 g), liver (˜1 g) and the left lower limb muscle(˜1 g) obtained at the completion of the study were weighed and thenhomogenized with 1 mL phosphate-buffered saline (PBS). Samples were thensonicated for 20 seconds for liver and lung samples or 60 seconds formuscle samples. Following centrifugation (14,000×g, 10 min, 22° C.), theresulting supernatants were tested immediately for MPO levels. The assaymixture consisted of o-dianasidine (2.85 mg/mL; Sigma), hydrogenperoxidase (0.85%) and a 1:40 dilution of samples in PBS. Absorbanceswere read at 450 nM, 5 min after substrate addition, and resultsexpressed as absorbance units/g tissue.

The level of MPO activity in the hindlimb muscles, lungs and liver ofrats were taken as a measurement of neutrophil sequestration into thetissue (Kyriakides et al., 2000). As shown in FIG. 17B, FIG. 17C andFIG. 17D, there were significant elevations in MPO activity in thehindlimb muscles, lungs and liver of I/R-only rats compared to those insham-operated rats (P>0.05), whereas sham-operated rats, ischemia-onlyrats had no significant increase in MPO activity in any of these tissues(P>0.05). Drug-treated rats in all 3 groups had significant decreases inMPO activity in all tissues compared to I/R-only rats (P<0.05), and thelevels were not significantly different from those in sham-operated rats(P>0.05).

Inhibition of Liver Homogenate TNF-αLevels of TNF-α; were measured inliver homogenate supernatant samples, using an enzyme-linkedimmunosorbent assay kit (OptEIA, Pharmingen, USA) as previouslydescribed (Strachan et al., 2000). A 1:10 dilution of supernatant fromliver homogenate samples was used in the assay. Supernatant was storedat −20° C., and samples were analyzed within 2 weeks of collection.Results were expressed as ng/g tissus. As shown in FIG. 18, liverhomogenate samples from I/R-only rats had significantly increasedTNF-αconcentrations compared to those from sham-operated rats (P<0.05).Levels of TNF-α in ischemia-only rats were not significantly differentfrom those from sham-operated rats (P>0.05). In drug-treated rats, all 3groups had a similar decrease in TNF-α concentrations compared toI/R-only rats (P<0.05), which were not significantly different fromthose from sham-operated animals (P>0.05).

Inhibition of Muscle Edema

Sections of the right lower limb muscle (˜1 g) obtained at thecompletion of the study were weighed and placed in an oven for 24 hoursat 80° C. before weighing again. The wet-to-dry weight ratio wasdetermined and taken as a measurement of muscle edema. As illustrated inFIG. 19, wet-to-dry weight ratios of the hindlimb muscle in I/R-onlyrats were significantly increased compared to those in sham-operatedanimals (P<0.05). Ratios in ischemia-only animals were not significantlydifferent from sham-operated animals (P>0.05). In all 3 groups ofdrug-treated rats, there was a similar decrease in wet-to-dry ratioscompared to those in I/R-only rats (P<0.05), and these values were notsignificantly different from those in sham-operated animals (P>0.05).

The results show that rats subjected to 2 hours of tourniquet-inducedbilateral hindlimb ischemia and 4 hours reperfusion suffered both localinjury and injury to the lungs, liver and kidney, as measured by variousindices of tissue stress. Rats subjected to ischemia alone had nosignificant alterations in disease markers compared to sham-operatedanimals. Blood taken from I/R-only animals after only 10 min ofreperfusion also had no significant changes in the plasma or serumlevels of CK, LDH, AST, ALT, K+ or Ca++ compared to sham-operatedanimals. The severity of local skeletal muscle injury was assessed bymeasuring increases in muscle edema and neutrophil accumulationfollowing 4 hours of reperfusion, as well as serum CK and LDH throughoutthe reperfusion period. The cytosolic enzyme CK is found predominantlyin muscle, and is-a reliable marker of muscle tissue damage (Tay et al.,2000). Lactate dehydrogenase is also a cytosolic enzyme found in themuscle, but is present in many other tissues as well (Carter et al.,1998). Consequently, LDH was a less specific measure of muscle injury,but still provided a measure of general tissue injury.

Indices of remote organ injury were detected in the lungs, liver andkidneys of animals subjected to ischemia and reperfusion episodes. Thepotential for lung injury was assessed by measuring increases inneutrophil accumulation in lung parenchyma. Hepatic injury was alsoquantified by measuring the increase in hepatic TNF-α and neutrophilaccumulation, and by measuring increases in plasma levels of ALT andAST. Although increases in plasma levels of ALT and AST have typicallybeen used as markers of liver pathology, both of these enzymes are alsofound within the muscle, and thus any increases may in part beattributed to muscle, rather than liver damage (Tay et al., 2000).Kidney dysfunction following skeletal muscle I/R is common (Tanaka etal., 1995). We found increases in serum BUN and plasma creatinine in I/Rrats. However, creatinine, and in particular BUN, are also derived fromthe muscle, and the observed increases may also be attributed to muscleinjury (Carter et al., 1998). We found a sizeable increase inproteinuria in I/R rats, indicating some degree of kidney injury.

The C5a antagonist AcF-[OPdChaWR] was found to inhibit a multitude ofdisease markers of local tissue and remote organ injury in this model.These results indicate a key role for C5a in the pathophysiology ofskeletal muscle I/R injury. Given the high incidence of complicationsfollowing lower limb ischemia and reperfusion in humans, the C5areceptor antagonists of the invention represent a possible futuretreatment of these complications, especially when I/R injury isanticipated, such as in surgical procedures. The ability of C5aantagonists to block both proinflammatory cytokine production andneutrophil trafficking may be key factors in their disease-modifyingproperties. The oral activity demonstrated here is a useful drugproperty for its widespread use in clinical situations.

DISCUSSION

This invention describes a series of conformationally-constrainedturn-containing cyclic molecules which are pre-organized for binding tocells which also bind human C5a. The principal feature of the compoundsof the invention is the pre-organized turn conformation presented by thecyclic scaffold, which assembles at least three hydrophobic groups intoneighbouring space, creating a hydrophobic surface ‘patch’.

This turn conformation of the antagonist may permit the cyclic peptideto bind in the transmembrane region of the C5a receptor at, or close to,the location which is also bound by the C-terminal end of human C5a.

The results described herein enable the design and development of evenmore potent conformationally constrained small molecule antagonists ofC5a. In principle the features of these cyclic antagonists are alsouseful for designing unrelated non-peptidic templates which similarlyproject substituents, corresponding to or similar to those attached tothe cyclic peptide scaffolds described herein, into similarthree-dimensional space as that occupied by these C5a receptorantagonists when bound to the receptor.

Cyclic peptides have several important advantages over acyclic peptidesas drug candidates (Fairlie et al., 1995, Fairlie et al., 1998, Tyndalland Fairlie, 2001). The cyclic compounds described in this specificationare stable to proteolytic degradation for at least several hours at 37°C. in human blood or plasma, in human or rat gastric juices, or inpresence of digestive enzymes such as pepsin, trypsin and chymotrypsin.In contrast, short linear peptides composed of L-amino acids are rapidlydegraded to their component amino acids within a few minutes under theseconditions. A second advantage lies in the constrained singleconformations adopted by the cyclic and non-peptidic molecules, incontrast to acyclic or linear peptides, which are flexible enough toadopt multiple structures in solution other than the one required forreceptor-binding. Thirdly, cyclic compounds such as those described inthis invention are usually more lipid-soluble and more pharmacologicallybioavailable as drugs than acyclic peptides, which can rarely beadministered orally. Fourthly, the plasma half-lives of cyclic moleculesare usually longer than those of peptides.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

REFERENCES

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1. A compound which is an antagonist of a C5a G protein-coupledreceptor, which has no C5a agonist activity, and which is a cyclicpeptide or peptidomimetic of the general formula:

wherein A is H, alkyl, aryl, NH-alkyl, N(alkyl)₂, NH-aryl, NH-benzoyl,NHSO₃, NHSO₂-alkyl, NHSO₂-aryl, OH, O-alkyl, or O-aryl; B is an alkyl,aryl, benzyl, naphthyl or indole group, or is the side chain ofL-phenylalanine or L-phenylglycine; C is a side chain of glycine,alanine, leucine, valine, proline, hydroxyproline, or thioproline; D isa side chain of D-leucine, D-homoleucine, D-cyclohexylalanine,D-homocyclohexylalanine, D-valine, D-norleucine, D-homo-norleucine,D-phenylalanine, D-tetrahydroisoquinoline, D-glutamine, D-glutamate, orD-tyrosine; E is L-1-napthyl or L-3-benzothienyl alanine, or is a sidechain of an amino acid selected from the group consisting ofL-phenylalanine, L-tryptophan and L-homotryptophan; F is a side chain ofL-arginine, L-homoarginine, L-citrulline, or L-canavanine; X is—(CH₂)_(n)NH— or (CH₂)_(n)—S—, where n is an integer of from 1 to 4;—(CH₂)₂O—; —(CH₂)₃O—; —(CH₂)₃—; —(CH₂)₄—; —CH₂COCHRNH—; or—CH₂—CHCOCHRNH—, and where R is a side chain of any common or uncommonamino acid, with the proviso that the compound is not AcF-[OPdChaWR](compound 1). 2-18. (canceled)
 19. The compound of claim 1, in which nis 2 or
 3. 20. The compound of claim 1, in which A is an aminomethylgroup, or a substituted or unsubstituted sulphonamide group.
 21. Thecompound of claim 20, in which A is a substituted sulfonamide group andwherein the substituent on the substituted sulfonamide is an alkyl chainof 1 to 6 carbon atoms, or a phenyl or toluoyl group.
 22. The compoundof claim 21, in which A is a substituted sulfonamide group, and whereinthe substituent on the substituted sulfonamide is an alkyl chain of 1 to4 carbon atoms.
 23. The compound of claim 1, in which the compound hasantagonist activity against a C5a receptor, a vasopressin receptor or aneurokinin receptor.
 24. The compound of claim 1, in which the compoundhas antagonist activity at submicromolar concentrations.
 25. Thecompound of claim 24, in which the compound has a receptor affinityIC₅₀<25 μM, and an antagonist potency IC₅₀<1 μM.
 26. A compound selectedfrom the group consisting of compounds 2, 10, 11 and
 17.


27. The compound of claim 1 or claim 26, together with apharmaceutically-acceptable carrier or excipient.
 28. The compound ofclaim 1, having the formula hydrocinnamate-[Orn-Pro-dCha-Trp-Arg].
 29. Acomposition comprising the compound of claim 1, and apharmaceutically-acceptable carrier or excipient.
 30. A method oftreatment of a pathological condition mediated by a G protein-coupledreceptor, comprising administering an effective amount of a compoundaccording to claim 1 to a mammal in need of such treatment.
 31. Themethod of claim 30, in which the condition mediated by a Gprotein-coupled receptor is a condition mediated by a C5a receptor. 32.The method of claim 31, in which the condition involves overexpressionor underregulation of C5a.
 33. The method of claim 32, in which thecondition comprises rheumatoid arthritis, adult respiratory distresssyndrome (ARDS), systemic lupus erythematosus, tissue graft rejection,ischemic heart disease, reperfusion injury, septic shock, gingivitis,fibrosis, atherosclerosis, multiple sclerosis, Alzheimer's disease,asthma, dementias, central nervous system disorders, lung injury,extracorporeal post-dialysis syndrome, or dermal inflammatory disorders.34. The method of claim 33, in which the condition is reperfusioninjury.
 35. A method of treating reperfusion injury, comprisingadministering to a mammal in need of such treatment an effective amountof a compound according to claim 1.